Publications
25.
Wenshu He, Stefan Burén, Can Baysal, Xi Jiang, Teresa Capell, Paul Christou, Luis M. Rubio
Nitrogenase cofactor maturase NifB isolated from transgenic rice is active in FeMo-co synthesis
Journal Article
ACS Synth Biol., vol. 11, iss. 9, pp. 3028–3036, 2022.
Abstract | Links:
@article{nokey,
title = {Nitrogenase cofactor maturase NifB isolated from transgenic rice is active in FeMo-co synthesis},
author = {Wenshu He and Stefan Burén and Can Baysal and Xi Jiang and Teresa Capell and Paul Christou and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9486962/pdf/sb2c00194.pdf},
doi = {10.1021/acssynbio.2c00194},
year = {2022},
date = {2022-09-16},
urldate = {2022-09-16},
journal = {ACS Synth Biol.},
volume = {11},
issue = {9},
pages = {3028–3036},
abstract = {The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe–9S–C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron–molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe–S] clusters are extremely unstable and sensitive to O_{2}. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea \emph{Methanocaldococcus infernus} and \emph{Methanothermobacter thermautotrophicus}. The recombinant proteins were targeted to the mitochondria to limit exposure to O_{2} and to have access to essential [4Fe–4S] clusters required for NifB-co biosynthesis. \emph{M. infernus} and \emph{M. thermautotrophicus} NifB accumulated as soluble proteins \emph{in planta}, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe–9S–C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron–molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe–S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe–4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.
24.
Lucía Payá-Tormo, Diana Coroian, Silvia Martín-Muñoz, Artavazd Badalyan, Robert T. Green, Marcel Veldhuizen, Xi Jiang, Gema López-Torrejón, Janneke Balk, Lance C. Seefeldt, Stefan Burén, Luis M. Rubio
A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation
Journal Article
Sci Rep., vol. 12, iss. 1, pp. 10367, 2022.
Abstract | Links:
@article{nokey,
title = {A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation},
author = {Lucía Payá-Tormo and Diana Coroian and Silvia Martín-Muñoz and Artavazd Badalyan and Robert T. Green and Marcel Veldhuizen and Xi Jiang and Gema López-Torrejón and Janneke Balk and Lance C. Seefeldt and Stefan Burén and Luis M. Rubio},
url = {https://www.nature.com/articles/s41598-022-14453-x},
doi = {10.1038/s41598-022-14453-x},
year = {2022},
date = {2022-06-20},
urldate = {2022-06-20},
journal = {Sci Rep.},
volume = {12},
issue = {1},
pages = {10367},
abstract = {Biological nitrogen fixation (BNF) is the reduction of N_{2} into NH_{3} in a group of prokaryotes by an extremely O_{2}-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S_{2}V^{red}) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S_{2}V^{red} to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O_{2} resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from \emph{Dehalococcoides ethenogenes} did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from \emph{Geobacter sulfurreducens} was more resistant to O_{2} exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O_{2} tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.
23.
Xi Jiang, Diana Coroian, Emma Barahona, Carlos Echavarri-Erasun, Rocío Castellanos-Rueda, Álvaro Eseverri, Jose A. Aznar-Moreno, Stefan Burén (corresponding author), Luis M. Rubio (corresponding author)
Functional nitrogenase cofactor maturase NifB in mitochondria and chloroplasts of Nicotiana benthamiana
Journal Article
mBio., vol. 13, iss. 3, 2022.
Abstract | Links:
@article{nokey,
title = {Functional nitrogenase cofactor maturase NifB in mitochondria and chloroplasts of \emph{Nicotiana benthamiana}},
author = {Xi Jiang and Diana Coroian and Emma Barahona and Carlos Echavarri-Erasun and Rocío Castellanos-Rueda and Álvaro Eseverri and Jose A. Aznar-Moreno and Stefan Burén (corresponding author) and Luis M. Rubio (corresponding author)},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239050/pdf/mbio.00268-22.pdf},
doi = {10.1128/mbio.00268-22},
year = {2022},
date = {2022-06-13},
urldate = {2022-06-13},
journal = {mBio.},
volume = {13},
issue = {3},
abstract = {Engineering plants to synthesize nitrogenase and assimilate atmospheric N_{2} will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct \emph{nifB} sequences originating from different phyla and ecological niches to restore diazotrophic growth of an \emph{Azotobacter vinelandii nifB} mutant. Twenty of these variants rescued the \emph{nifB} mutant phenotype despite their phylogenetic distance to \emph{A. vinelandii}. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, \emph{nifB} functional modules composed of a \emph{nifB} variant, together with the \emph{A. vinelandii} NifS and NifU proteins (for biosynthesis of NifB [Fe_{4}S_{4}] clusters) and the FdxN ferredoxin (for NifB function), were expressed in \emph{Nicotiana benthamiana} chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (\emph{Methanosarcina acetivorans} and \emph{Methanocaldococcus infernus}) or mitochondria (\emph{M. infernus} and \emph{Methanothermobacter thermautotrophicus}). These NifB proteins were shown to accept [Fe_{4}S_{4}] clusters from NifU and were functional in FeMo-co synthesis \emph{in vitro}. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants.
22.
Casey Van Stappen, Emilio Jiménez-Vicente, Ana Pérez-González, Zhi-Yong Yang, Lance C. Seefeldt, Serena DeBeer, Dennis R. Dean, Laure Decamps
A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster
Journal Article
Chem. Sci., vol. 13, iss. 12, pp. 3489-3500, 2022.
Abstract | Links:
@article{nokey,
title = {A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster},
author = {Casey Van Stappen and Emilio Jiménez-Vicente and Ana Pérez-González and Zhi-Yong Yang and Lance C. Seefeldt and Serena DeBeer and Dennis R. Dean and Laure Decamps},
url = {https://pubs.rsc.org/en/content/articlepdf/2022/sc/d1sc06418e},
doi = {10.1039/D1SC06418E},
year = {2022},
date = {2022-02-28},
urldate = {2022-02-28},
journal = {Chem. Sci.},
volume = {13},
issue = {12},
pages = {3489-3500},
publisher = {The Royal Society of Chemistry},
abstract = {Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD_{2}K_{2} polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD_{2}K_{2}. P-cluster maturation involves the iron protein NifH_{2} as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD_{2}K_{2} species bearing immature P-clusters were isolated from an \emph{Azotobacter vinelandii} strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD_{2}K_{2} complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]^{2+/+} clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.
21.
Ana Pérez-González, Emilio Jimenez-Vicente, Alvaro Salinero-Lanzarote, Derek F. Harris, Lance C. Seefeldt, Dennis R. Dean
AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor
Journal Article
Mol. Microbiol., vol. 117, iss. 5, pp. 1080-1088, 2022.
Abstract | Links:
@article{nokey,
title = {AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor},
author = {Ana Pérez-González and Emilio Jimenez-Vicente and Alvaro Salinero-Lanzarote and Derek F. Harris and Lance C. Seefeldt and Dennis R. Dean},
url = {https://onlinelibrary.wiley.com/doi/epdf/10.1111/mmi.14890},
doi = {10.1111/mmi.14890 },
year = {2022},
date = {2022-02-27},
urldate = {2022-02-27},
journal = {Mol. Microbiol.},
volume = {117},
issue = {5},
pages = {1080-1088},
abstract = {\emph{Azotobacter vinelandii} produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N_{2} binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N_{2} fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N_{2}. These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2. These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.
20.
Ana Pérez-González, Emilio Jimenez-Vicente, Jakob Gies-Elterlein, Alvaro Salinero-Lanzarote, Zhi-Yong Yang, Oliver Einsle, Lance C. Seefeldt, Dennis R. Dean
Specificity of NifEN and VnfEN for the assembly of nitrogenase active site cofactors in Azotobacter vinelandii
Journal Article
mBio., vol. 12, iss. 4, 2021.
Abstract | Links:
@article{nokey,
title = {Specificity of NifEN and VnfEN for the assembly of nitrogenase active site cofactors in \emph{Azotobacter vinelandii}},
author = {Ana Pérez-González and Emilio Jimenez-Vicente and Jakob Gies-Elterlein and Alvaro Salinero-Lanzarote and Zhi-Yong Yang and Oliver Einsle and Lance C. Seefeldt and Dennis R. Dean},
url = {https://journals.asm.org/doi/reader/10.1128/mBio.01568-21},
doi = {10.1128/mBio.01568-21},
year = {2021},
date = {2021-08-31},
urldate = {2021-08-31},
journal = {mBio.},
volume = {12},
issue = {4},
abstract = {The nitrogen-fixing microbe \emph{Azotobacter vinelandii} has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their \emph{in vivo} cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in \emph{A. vinelandii} that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site.
\textbf{IMPORTANCE}: Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site.
IMPORTANCE: Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.
19.
Emma Barahona, Xi Jiang, Emilio Jiménez-Vicente, Luis M. Rubio, Manuel González-Guerrero
Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase
Journal Article Forthcoming
bioRxiv, Forthcoming.
Abstract | Links:
@article{nokey,
title = {\emph{Azotobacter vinelandii} scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase},
author = {Emma Barahona and Xi Jiang and Emilio Jiménez-Vicente and Luis M. Rubio and Manuel González-Guerrero},
url = {https://www.biorxiv.org/content/10.1101/2021.12.06.471366v1.full.pdf},
doi = {10.1101/2021.12.06.471366},
year = {2021},
date = {2021-06-12},
urldate = {2021-06-12},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {\emph{Azotobacter vinelandii} molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe_{3}S_{4}] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe_{4}-S_{4}] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.},
keywords = {},
pubstate = {forthcoming},
tppubtype = {article}
}
Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.
18.
Gema López-Torrejón, Stefan Burén, Marcel Veldhuizen, Luis M. Rubio
Biosynthesis of cofactor‐activatable iron‐only nitrogenase in Saccharomyces cerevisiae
Journal Article
Microb Biotechnol., vol. 14, iss. 3, pp. 1073-1083, 2021.
Abstract | Links:
@article{nokey,
title = {Biosynthesis of cofactor‐activatable iron‐only nitrogenase in \emph{Saccharomyces cerevisiae}},
author = {Gema López-Torrejón and Stefan Burén and Marcel Veldhuizen and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085987/pdf/MBT2-14-1073.pdf},
doi = {10.1111/1751-7915.13758},
year = {2021},
date = {2021-05-25},
urldate = {2021-05-25},
journal = {Microb Biotechnol.},
volume = {14},
issue = {3},
pages = {1073-1083},
abstract = {Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe‐only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe‐only nitrogenase component proteins in the low‐oxygen mitochondria matrix of \emph{S. cerevisiae}. As‐isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo‐AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo‐AnfDK could be activated for acetylene reduction by the simple addition of FeMo‐co \emph{in vitro}, indicating preexistence of the P‐clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe‐only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.
Fe‐only nitrogenase components were expressed and matured in \emph{S. cerevisiae} mitochondrial. The electron donor component was active as isolated and the catalytic component was ready for activation by cofactor insertion.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe‐only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe‐only nitrogenase component proteins in the low‐oxygen mitochondria matrix of S. cerevisiae. As‐isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo‐AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo‐AnfDK could be activated for acetylene reduction by the simple addition of FeMo‐co in vitro, indicating preexistence of the P‐clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe‐only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.
Fe‐only nitrogenase components were expressed and matured in S. cerevisiae mitochondrial. The electron donor component was active as isolated and the catalytic component was ready for activation by cofactor insertion.
17.
Xi Jiang, Lucía Payá-Tormo, Diana Coroian, Inés García-Rubio, Rocío Castellanos-Rueda, Álvaro Eseverri, Gema López-Torrejón, Stefan Burén (corresponding author), Luis M. Rubio
Commun Biol., vol. 4, iss. 1, pp. 4, 2021.
Abstract | Links:
@article{nokey,
title = {Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N_{2}-fixation in plants},
author = {Xi Jiang and Lucía Payá-Tormo and Diana Coroian and Inés García-Rubio and Rocío Castellanos-Rueda and Álvaro Eseverri and Gema López-Torrejón and Stefan Burén (corresponding author) and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782807/pdf/42003_2020_Article_1536.pdf},
doi = {10.1038/s42003-020-01536-6},
year = {2021},
date = {2021-01-04},
urldate = {2021-01-04},
journal = {Commun Biol.},
volume = {4},
issue = {1},
pages = {4},
abstract = {Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase \emph{nifH} sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. \emph{Hydrogenobacter thermophilus} (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N_{2} fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. \emph{H. thermophilus} NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe_{4}S_{4}] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.
16.
Zhi-Yong Yang, Emilio Jimenez-Vicente, Hayden Kallas, Dmitriy A. Lukoyanov, Hao Yang, Julia S. Martin del Campo, Dennis R. Dean, Brian M. Hoffman, Lance C. Seefeldt
The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase
Journal Article
Chem. Sci., vol. 12, iss. 20, pp. 6913-6922, 2021.
Abstract | Links:
@article{nokey,
title = {The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase},
author = {Zhi-Yong Yang and Emilio Jimenez-Vicente and Hayden Kallas and Dmitriy A. Lukoyanov and Hao Yang and Julia S. Martin del Campo and Dennis R. Dean and Brian M. Hoffman and Lance C. Seefeldt},
url = {https://pubs.rsc.org/en/content/articlepdf/2021/sc/d0sc06561g},
doi = {10.1039/D0SC06561G},
year = {2021},
date = {2021-03-29},
urldate = {2021-03-29},
journal = {Chem. Sci.},
volume = {12},
issue = {20},
pages = {6913-6922},
publisher = {The Royal Society of Chemistry},
abstract = {The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad \emph{S} = 3/2 EPR signal (Kramers state) having \emph{g} values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V^{3+}, 3Fe^{3+}, 4Fe^{2+}]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, \emph{S} = 0, or non-Kramers, integer-spin (\emph{S} = 1, 2 \emph{etc.}). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic \emph{S} = 1/2 EPR signal from such a reduced state is observed, with \emph{g} = [2.18, 2.12, 2.09] and showing well-defined ^{51}V (\emph{I} = 7/2) hyperfine splitting, \emph{a}_{iso} = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: \emph{S} = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V^{3+}, 4Fe^{3+}, 3Fe^{2+}]. Our findings suggest that the V^{3+} does not change valency throughout the catalytic cycle.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V<small>3+</small>, 3Fe<small>3+</small>, 4Fe<small>2+</small>]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined <small>51</small>V (I = 7/2) hyperfine splitting, a<small>iso</small> = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V<small>3+</small>, 4Fe<small>3+</small>, 3Fe<small>2+</small>]. Our findings suggest that the V<small>3+</small> does not change valency throughout the catalytic cycle.
15.
Álvaro Eseverri, Can Baysal, Vicente Medina, Teresa Capell, Paul Christou, Luis M. Rubio, Elena Caro
Front. Plant Sci., vol. 11, 2020, ISSN: 1664-462X.
Abstract | Links:
@article{nokey,
title = {Transit peptides from photosynthesis-related proteins mediate import of a marker protein into different plastid types and within different species},
author = {Álvaro Eseverri and Can Baysal and Vicente Medina and Teresa Capell and Paul Christou and Luis M. Rubio and Elena Caro},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2020.560701/pdf},
doi = {10.3389/fpls.2020.560701},
issn = {1664-462X},
year = {2020},
date = {2020-09-25},
urldate = {2020-09-25},
journal = {Front. Plant Sci.},
volume = {11},
abstract = {Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four \emph{Arabidopsis thaliana} proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In \emph{A. thaliana}, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.
14.
Álvaro Eseverri, Gema López-Torrejón, Xi Jiang, Stefan Burén, Luis M. Rubio, Elena Caro
Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells
Journal Article
Plant Biotechnol J., vol. 18, no. 9, pp. 1882-1896, 2020.
Abstract | Links:
@article{nokey,
title = {Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells},
author = {Álvaro Eseverri and Gema López-Torrejón and Xi Jiang and Stefan Burén and Luis M. Rubio and Elena Caro},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.13347},
doi = {10.1111/pbi.13347},
year = {2020},
date = {2020-01-27},
journal = {Plant Biotechnol J.},
volume = {18},
number = {9},
pages = {1882-1896},
abstract = {The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. \emph{Azotobacter vinelandii} nitrogen fixation genes, \emph{nifH}, \emph{M}, \emph{U} and \emph{S,} were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of \emph{n}\emph{ifU} and \emph{n}\emph{ifS} co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.
13.
Stefan Burén, Emilio Jiménez-Vicente, Carlos Echavarri-Erasun, Luis M. Rubio
Biosynthesis of nitrogenase cofactors
Journal Article
Chem. Rev., vol. 120, iss. 12, pp. 4921–4968, 2020, ISSN: 0009-2665.
Abstract | Links:
@article{nokey,
title = {Biosynthesis of nitrogenase cofactors},
author = {Stefan Burén and Emilio Jiménez-Vicente and Carlos Echavarri-Erasun and Luis M. Rubio},
url = {https://doi.org/10.1021/acs.chemrev.9b00489},
doi = {10.1021/acs.chemrev.9b00489},
issn = {0009-2665},
year = {2020},
date = {2020-06-24},
urldate = {2020-06-24},
journal = {Chem. Rev.},
volume = {120},
issue = {12},
pages = {4921–4968},
publisher = {American Chemical Society},
abstract = {Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo-\emph{R}-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both \emph{in situ}, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo-R-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both in situ, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.
12.
Stefan Burén, Katelin Pratt, Xi Jiang, Yisong Guo, Emilio Jimenez-Vicente, Carlos Echavarri-Erasun, Dennis R. Dean, Ishtiaq Saaem, D. Benjamin Gordon, Christopher A. Voigt, Luis M. Rubio
Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae
Journal Article
PNAS, vol. 116, no. 50, pp. 25078-25086, 2019.
Abstract | Links:
@article{nokey,
title = {Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in \emph{Saccharomyces cerevisiae}},
author = {Stefan Burén and Katelin Pratt and Xi Jiang and Yisong Guo and Emilio Jimenez-Vicente and Carlos Echavarri-Erasun and Dennis R. Dean and Ishtiaq Saaem and D. Benjamin Gordon and Christopher A. Voigt and Luis M. Rubio},
url = {https://www.pnas.org/doi/abs/10.1073/pnas.1904903116},
doi = {10.1073/pnas.1904903116},
year = {2019},
date = {2019-11-25},
journal = {PNAS},
volume = {116},
number = {50},
pages = {25078-25086},
abstract = {The radical \textit{S}-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured \textit{Saccharomyces cerevisiae}. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted \textit{nif} genes (\emph{nifUSXB} and \emph{fdxN}) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including \emph{nifUSX} and \emph{fdxN}, and a library consisting of 28 different \emph{nifB} genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from \emph{Azotobacter vinelandii} with NifB from the archaea \emph{Methanocaldococcus infernus} or \emph{Methanothermobacter thermautotrophicus} yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured \emph{S. cerevisiae} coexpressing \emph{M. thermautotrophicus} NifB with \emph{A. vinelandii} NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.
11.
Can Baysal, Ana Pérez-González, Álvaro Eseverri, Xi Jiang, Vicente Medina, Elena Caro, Luis M. Rubio, Paul Christou, Changfu Zhu
Transgenic Res., vol. 29, pp. 37-52, 2019.
Abstract | Links:
@article{nokey,
title = {Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria},
author = {Can Baysal and Ana Pérez-González and Álvaro Eseverri and Xi Jiang and Vicente Medina and Elena Caro and Luis M. Rubio and Paul Christou and Changfu Zhu},
url = {https://link.springer.com/article/10.1007/s11248-019-00176-9},
doi = {10.1007/s11248-019-00176-9},
year = {2019},
date = {2019-10-10},
journal = {Transgenic Res.},
volume = {29},
pages = {37-52},
abstract = {Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (\emph{Oryza sativa}). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (\emph{Saccharomyces cerevisiae}), SU9 (\emph{Neurospora crassa}), pFA (\emph{Arabidopsis thaliana}) and OsSCSb (\emph{Oryza sativa}) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (\emph{Nicotiana plumbaginifolia}) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.
10.
Mónica Navarro-Rodríguez, José María Buesa, Luis M. Rubio
Genetic and biochemical analysis of the Azotobacter vinelandii molybdenum storage protein
Journal Article
Front. Microbiol., vol. 10, pp. 579, 2019.
Abstract | Links:
@article{nokey,
title = {Genetic and biochemical analysis of the \emph{Azotobacter vinelandii} molybdenum storage protein},
author = {Mónica Navarro-Rodríguez and José María Buesa and Luis M. Rubio},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2019.00579/full},
doi = {10.3389/fmicb.2019.00579},
year = {2019},
date = {2019-03-21},
urldate = {2019-03-21},
journal = {Front. Microbiol.},
volume = {10},
pages = {579},
abstract = {The N_{2} fixing bacterium \emph{Azotobacter vinelandii} carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The \emph{in vitro} FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an \emph{A. vinelandii} strain lacking the MoSto subunit genes (Δ\emph{mosAB}) was analyzed. Consistent with its role as storage protein, the Δ\emph{mosAB} strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased \emph{in vivo} nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The Δ\emph{mosAB} strain was found to readily derepress \emph{vnfDGK} upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The Δ\emph{mosAB} mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δ\emph{vnf} Δ\emph{anf}) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the Δ\emph{mosAB} and \emph{mosAB}^{+} strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to \emph{A. vinelandii} in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (ΔmosAB) was analyzed. Consistent with its role as storage protein, the ΔmosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The ΔmosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The ΔmosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δvnf Δanf) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the ΔmosAB and mosAB+ strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.
9.
Emilio Jimenez-Vicente, Zhi-Yong Yang, Julia S. Martin del Campo, Valerie L. Cash, Lance C. Seefeldt, Dennis R. Dean
The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters
Journal Article
J. Biol. Chem., vol. 294, iss. 16, pp. 6204-6213, 2019.
Abstract | Links:
@article{nokey,
title = {The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters},
author = {Emilio Jimenez-Vicente and Zhi-Yong Yang and Julia S. Martin del Campo and Valerie L. Cash and Lance C. Seefeldt and Dennis R. Dean},
doi = {10.1074/jbc.RA119.007905},
year = {2019},
date = {2019-04-19},
urldate = {2019-04-19},
journal = {J. Biol. Chem.},
volume = {294},
issue = {16},
pages = {6204-6213},
abstract = {The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α_{2}β_{2} heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N_{2} reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N_{2}-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as \emph{in vivo}, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe \emph{Azotobacter vinelandii}. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature \emph{in vitro} without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α2β2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein’s catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.
8.
Stefan Burén, Gema López-Torrejón, Luis M. Rubio
Extreme bioengineering to meet the nitrogen challenge
Journal Article
PNAS, vol. 115, no. 36, pp. 8849-8851, 2018.
@article{nokey,
title = {Extreme bioengineering to meet the nitrogen challenge},
author = {Stefan Burén and Gema López-Torrejón and Luis M. Rubio},
url = {https://www.pnas.org/doi/abs/10.1073/pnas.1812247115},
doi = {10.1073/pnas.1812247115},
year = {2018},
date = {2018-08-16},
journal = {PNAS},
volume = {115},
number = {36},
pages = {8849-8851},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
7.
Emilio Jimenez-Vicente, Zhi-Yong Yang, W. Keith Ray, Carlos Echavarri-Erasun, Valerie L. Cash, Luis M. Rubio, Lance C. Seefeldt, Dennis R. Dean
Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
Journal Article
J. Biol. Chem., vol. 293, iss. 25, pp. 9812-9823, 2018, ISSN: 0021-9258.
Abstract | Links:
@article{nokey,
title = {Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation},
author = {Emilio Jimenez-Vicente and Zhi-Yong Yang and W. Keith Ray and Carlos Echavarri-Erasun and Valerie L. Cash and Luis M. Rubio and Lance C. Seefeldt and Dennis R. Dean},
url = {https://www.sciencedirect.com/science/article/pii/S0021925820399658},
doi = {10.1074/jbc.RA118.002994},
issn = {0021-9258},
year = {2018},
date = {2018-06-12},
urldate = {2018-06-12},
journal = {J. Biol. Chem.},
volume = {293},
issue = {25},
pages = {9812-9823},
abstract = {Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium \emph{Azotobacter vinelandii}. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an \emph{A. vinelandii} strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
6.
Stefan Burén, Luis M. Rubio
State of the art in eukaryotic nitrogenase engineering
Journal Article
FEMS Microbiol. Lett., vol. 365, iss. 2, 2017, ISSN: 0378-1097.
Abstract | Links:
@article{nokey,
title = {State of the art in eukaryotic nitrogenase engineering},
author = {Stefan Burén and Luis M. Rubio},
url = {https://doi.org/10.1093/femsle/fnx274},
doi = {10.1093/femsle/fnx274},
issn = {0378-1097},
year = {2017},
date = {2017-12-12},
journal = {FEMS Microbiol. Lett.},
volume = {365},
issue = {2},
abstract = {Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.
5.
Simon Arragain, Emilio Jiménez-Vicente, Alessandro A. Scandurra, Stefan Burén, Luis M. Rubio, Carlos Echavarri-Erasun
Diversity and functional analysis of the FeMo-cofactor maturase NifB
Journal Article
Front. Plant Sci., vol. 8, 2017, ISSN: 1664-462X.
Abstract | Links:
@article{nokey,
title = {Diversity and functional analysis of the FeMo-cofactor maturase NifB},
author = {Simon Arragain and Emilio Jiménez-Vicente and Alessandro A. Scandurra and Stefan Burén and Luis M. Rubio and Carlos Echavarri-Erasun},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2017.01947/pdf},
doi = {10.3389/fpls.2017.01947},
issn = {1664-462X},
year = {2017},
date = {2017-11-14},
urldate = {2017-11-14},
journal = {Front. Plant Sci.},
volume = {8},
abstract = {One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile \emph{Chlorobium tepidum} (two-domain architecture), the hyperthermophile \emph{Methanocaldococcus infernus} (single-domain architecture) and the mesophile \emph{Klebsiella oxytoca} (two-domain architecture), showing \emph{in silico} characterization of their nitrogen fixation (\emph{nif}) gene clusters, conserved NifB motifs, and functionality. \emph{C. tepidum} and \emph{M. infernus} NifB were able to complement an \emph{Azotobacter vinelandii} (Δ\emph{nifB}) mutant restoring the Nif^{+} phenotype and thus demonstrating their functionality \emph{in vivo}. In addition, purified \emph{C. tepidum} NifB exhibited activity in the \emph{in vitro} NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain \emph{K. oxytoca} NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher \emph{in vivo} nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
4.
Ana Pérez-González, Ryan Kniewel, Marcel Veldhuizen, Hemant K. Verma, Mónica Navarro-Rodríguez, Luis M. Rubio, Elena Caro
BMC Biotechnol., vol. 17, no. 80, 2017, ISSN: 1472-6750.
Abstract | Links:
@article{nokey,
title = {Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria},
author = {Ana Pérez-González and Ryan Kniewel and Marcel Veldhuizen and Hemant K. Verma and Mónica Navarro-Rodríguez and Luis M. Rubio and Elena Caro},
url = {https://doi.org/10.1186/s12896-017-0393-y},
doi = {10.1186/s12896-017-0393-y},
issn = {1472-6750},
year = {2017},
date = {2017-11-13},
urldate = {2017-11-13},
journal = {BMC Biotechnol.},
volume = {17},
number = {80},
abstract = {\textbf{Background}
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
\textbf{Results}
Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within \emph{Saccharomyces cerevisiae}. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (\emph{nif}) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, \emph{nifU}, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.
\textbf{Conclusions}
We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Background
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
Results
Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.
Conclusions
We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.
3.
Stefan Burén, Xi Jiang, Gema López-Torrejón, Carlos Echavarri-Erasun, Luis M. Rubio
Purification and in vitro activity of mitochondria targeted nitrogenase cofactor maturase NifB
Journal Article
Front. Plant Sci., vol. 8, 2017, ISSN: 1664-462X.
Abstract | Links:
@article{nokey,
title = {Purification and \emph{in vitro} activity of mitochondria targeted nitrogenase cofactor maturase NifB},
author = {Stefan Burén and Xi Jiang and Gema López-Torrejón and Carlos Echavarri-Erasun and Luis M. Rubio},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2017.01567/full},
doi = {10.3389/fpls.2017.01567},
issn = {1664-462X},
year = {2017},
date = {2017-09-12},
urldate = {2017-09-12},
journal = {Front. Plant Sci.},
volume = {8},
abstract = {Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O_{2}-sensitive \emph{S}-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium \emph{Azotobacter vinelandii} and the thermophilic methanogen \emph{Methanocaldococcus infernus} in \emph{Saccharomyces cerevisiae} and in \emph{Nicotiana benthamiana}. NifB proteins were targeted to the mitochondria, where O_{2} consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the \emph{A. vinelandii} version appeared to form aggregates. Similarly, NifB from \emph{M. infernus} was expressed at higher levels in leaves of \emph{Nicotiana benthamiana} and accumulated as a soluble protein while \emph{A. vinelandii} NifB was mainly associated with the non-soluble cell fraction. Soluble \emph{M. infernus} NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the \emph{in vitro} FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening \emph{nif} genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.
2.
Stefan Burén, Eric M. Young, Elizabeth A. Sweeny, Gema Lopez-Torrejón, Marcel Veldhuizen, Christopher A. Voigt, Luis M. Rubio
Formation of nitrogenase NifDK tetramers in the mitochondria of Saccharomyces cerevisiae
Journal Article
ACS Synth. Biol., vol. 6, iss. 6, pp. 1043–1055, 2017.
Abstract | Links:
@article{nokey,
title = {Formation of nitrogenase NifDK tetramers in the mitochondria of \emph{Saccharomyces cerevisiae}},
author = {Stefan Burén and Eric M. Young and Elizabeth A. Sweeny and Gema Lopez-Torrejón and Marcel Veldhuizen and Christopher A. Voigt and Luis M. Rubio},
url = {https://doi.org/10.1021/acssynbio.6b00371},
doi = {10.1021/acssynbio.6b00371},
year = {2017},
date = {2017-02-21},
urldate = {2017-02-21},
journal = {ACS Synth. Biol.},
volume = {6},
issue = {6},
pages = {1043–1055},
abstract = {Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N_{2} fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O_{2} consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of \emph{Saccharomyces cerevisiae} in which transcriptional units comprising nine \emph{Azotobacter vinelandii nif} genes (\emph{nifHDKUSMBEN}) were integrated into the genome. Two combinatorial libraries of \emph{nif} gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of \emph{nif} gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and \emph{nif} expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust \emph{nif} gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.
1.
Gema López-Torrejón, Emilio Jiménez-Vicente, José María Buesa, Jose A. Hernandez, Hemant K. Verma, Luis M. Rubio
Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast
Journal Article
Nat. Commun., vol. 7, iss. 1, pp. 11426, 2016.
Abstract | Links:
@article{nokey,
title = {Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast},
author = {Gema López-Torrejón and Emilio Jiménez-Vicente and José María Buesa and Jose A. Hernandez and Hemant K. Verma and Luis M. Rubio},
url = {https://doi.org/10.1038/ncomms11426},
doi = {10.1038/ncomms11426},
year = {2016},
date = {2016-04-29},
journal = {Nat. Commun.},
volume = {7},
issue = {1},
pages = {11426},
abstract = {The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct \emph{nif} gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N_{2}-fixing cereals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.
2022
25.
Wenshu He, Stefan Burén, Can Baysal, Xi Jiang, Teresa Capell, Paul Christou, Luis M. Rubio
Nitrogenase cofactor maturase NifB isolated from transgenic rice is active in FeMo-co synthesis
ACS Synth Biol., vol. 11, iss. 9, pp. 3028–3036, 2022.
@article{nokey,
title = {Nitrogenase cofactor maturase NifB isolated from transgenic rice is active in FeMo-co synthesis},
author = {Wenshu He and Stefan Burén and Can Baysal and Xi Jiang and Teresa Capell and Paul Christou and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9486962/pdf/sb2c00194.pdf},
doi = {10.1021/acssynbio.2c00194},
year = {2022},
date = {2022-09-16},
urldate = {2022-09-16},
journal = {ACS Synth Biol.},
volume = {11},
issue = {9},
pages = {3028–3036},
abstract = {The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe–9S–C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron–molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe–S] clusters are extremely unstable and sensitive to O_{2}. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea \emph{Methanocaldococcus infernus} and \emph{Methanothermobacter thermautotrophicus}. The recombinant proteins were targeted to the mitochondria to limit exposure to O_{2} and to have access to essential [4Fe–4S] clusters required for NifB-co biosynthesis. \emph{M. infernus} and \emph{M. thermautotrophicus} NifB accumulated as soluble proteins \emph{in planta}, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe–9S–C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron–molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe–S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe–4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.
24.
Lucía Payá-Tormo, Diana Coroian, Silvia Martín-Muñoz, Artavazd Badalyan, Robert T. Green, Marcel Veldhuizen, Xi Jiang, Gema López-Torrejón, Janneke Balk, Lance C. Seefeldt, Stefan Burén, Luis M. Rubio
Sci Rep., vol. 12, iss. 1, pp. 10367, 2022.
@article{nokey,
title = {A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation},
author = {Lucía Payá-Tormo and Diana Coroian and Silvia Martín-Muñoz and Artavazd Badalyan and Robert T. Green and Marcel Veldhuizen and Xi Jiang and Gema López-Torrejón and Janneke Balk and Lance C. Seefeldt and Stefan Burén and Luis M. Rubio},
url = {https://www.nature.com/articles/s41598-022-14453-x},
doi = {10.1038/s41598-022-14453-x},
year = {2022},
date = {2022-06-20},
urldate = {2022-06-20},
journal = {Sci Rep.},
volume = {12},
issue = {1},
pages = {10367},
abstract = {Biological nitrogen fixation (BNF) is the reduction of N_{2} into NH_{3} in a group of prokaryotes by an extremely O_{2}-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S_{2}V^{red}) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S_{2}V^{red} to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O_{2} resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from \emph{Dehalococcoides ethenogenes} did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from \emph{Geobacter sulfurreducens} was more resistant to O_{2} exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O_{2} tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.
23.
Xi Jiang, Diana Coroian, Emma Barahona, Carlos Echavarri-Erasun, Rocío Castellanos-Rueda, Álvaro Eseverri, Jose A. Aznar-Moreno, Stefan Burén (corresponding author), Luis M. Rubio (corresponding author)
@article{nokey,
title = {Functional nitrogenase cofactor maturase NifB in mitochondria and chloroplasts of \emph{Nicotiana benthamiana}},
author = {Xi Jiang and Diana Coroian and Emma Barahona and Carlos Echavarri-Erasun and Rocío Castellanos-Rueda and Álvaro Eseverri and Jose A. Aznar-Moreno and Stefan Burén (corresponding author) and Luis M. Rubio (corresponding author)},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239050/pdf/mbio.00268-22.pdf},
doi = {10.1128/mbio.00268-22},
year = {2022},
date = {2022-06-13},
urldate = {2022-06-13},
journal = {mBio.},
volume = {13},
issue = {3},
abstract = {Engineering plants to synthesize nitrogenase and assimilate atmospheric N_{2} will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct \emph{nifB} sequences originating from different phyla and ecological niches to restore diazotrophic growth of an \emph{Azotobacter vinelandii nifB} mutant. Twenty of these variants rescued the \emph{nifB} mutant phenotype despite their phylogenetic distance to \emph{A. vinelandii}. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, \emph{nifB} functional modules composed of a \emph{nifB} variant, together with the \emph{A. vinelandii} NifS and NifU proteins (for biosynthesis of NifB [Fe_{4}S_{4}] clusters) and the FdxN ferredoxin (for NifB function), were expressed in \emph{Nicotiana benthamiana} chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (\emph{Methanosarcina acetivorans} and \emph{Methanocaldococcus infernus}) or mitochondria (\emph{M. infernus} and \emph{Methanothermobacter thermautotrophicus}). These NifB proteins were shown to accept [Fe_{4}S_{4}] clusters from NifU and were functional in FeMo-co synthesis \emph{in vitro}. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants.
22.
Casey Van Stappen, Emilio Jiménez-Vicente, Ana Pérez-González, Zhi-Yong Yang, Lance C. Seefeldt, Serena DeBeer, Dennis R. Dean, Laure Decamps
A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster
Chem. Sci., vol. 13, iss. 12, pp. 3489-3500, 2022.
@article{nokey,
title = {A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster},
author = {Casey Van Stappen and Emilio Jiménez-Vicente and Ana Pérez-González and Zhi-Yong Yang and Lance C. Seefeldt and Serena DeBeer and Dennis R. Dean and Laure Decamps},
url = {https://pubs.rsc.org/en/content/articlepdf/2022/sc/d1sc06418e},
doi = {10.1039/D1SC06418E},
year = {2022},
date = {2022-02-28},
urldate = {2022-02-28},
journal = {Chem. Sci.},
volume = {13},
issue = {12},
pages = {3489-3500},
publisher = {The Royal Society of Chemistry},
abstract = {Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD_{2}K_{2} polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD_{2}K_{2}. P-cluster maturation involves the iron protein NifH_{2} as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD_{2}K_{2} species bearing immature P-clusters were isolated from an \emph{Azotobacter vinelandii} strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD_{2}K_{2} complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]^{2+/+} clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.
21.
Ana Pérez-González, Emilio Jimenez-Vicente, Alvaro Salinero-Lanzarote, Derek F. Harris, Lance C. Seefeldt, Dennis R. Dean
Mol. Microbiol., vol. 117, iss. 5, pp. 1080-1088, 2022.
@article{nokey,
title = {AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor},
author = {Ana Pérez-González and Emilio Jimenez-Vicente and Alvaro Salinero-Lanzarote and Derek F. Harris and Lance C. Seefeldt and Dennis R. Dean},
url = {https://onlinelibrary.wiley.com/doi/epdf/10.1111/mmi.14890},
doi = {10.1111/mmi.14890 },
year = {2022},
date = {2022-02-27},
urldate = {2022-02-27},
journal = {Mol. Microbiol.},
volume = {117},
issue = {5},
pages = {1080-1088},
abstract = {\emph{Azotobacter vinelandii} produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N_{2} binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N_{2} fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N_{2}. These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2. These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.
2021
20.
Ana Pérez-González, Emilio Jimenez-Vicente, Jakob Gies-Elterlein, Alvaro Salinero-Lanzarote, Zhi-Yong Yang, Oliver Einsle, Lance C. Seefeldt, Dennis R. Dean
@article{nokey,
title = {Specificity of NifEN and VnfEN for the assembly of nitrogenase active site cofactors in \emph{Azotobacter vinelandii}},
author = {Ana Pérez-González and Emilio Jimenez-Vicente and Jakob Gies-Elterlein and Alvaro Salinero-Lanzarote and Zhi-Yong Yang and Oliver Einsle and Lance C. Seefeldt and Dennis R. Dean},
url = {https://journals.asm.org/doi/reader/10.1128/mBio.01568-21},
doi = {10.1128/mBio.01568-21},
year = {2021},
date = {2021-08-31},
urldate = {2021-08-31},
journal = {mBio.},
volume = {12},
issue = {4},
abstract = {The nitrogen-fixing microbe \emph{Azotobacter vinelandii} has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their \emph{in vivo} cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in \emph{A. vinelandii} that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site.
\textbf{IMPORTANCE}: Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site.
IMPORTANCE: Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.
19.
Emma Barahona, Xi Jiang, Emilio Jiménez-Vicente, Luis M. Rubio, Manuel González-Guerrero
@article{nokey,
title = {\emph{Azotobacter vinelandii} scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase},
author = {Emma Barahona and Xi Jiang and Emilio Jiménez-Vicente and Luis M. Rubio and Manuel González-Guerrero},
url = {https://www.biorxiv.org/content/10.1101/2021.12.06.471366v1.full.pdf},
doi = {10.1101/2021.12.06.471366},
year = {2021},
date = {2021-06-12},
urldate = {2021-06-12},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {\emph{Azotobacter vinelandii} molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe_{3}S_{4}] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe_{4}-S_{4}] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.},
keywords = {},
pubstate = {forthcoming},
tppubtype = {article}
}
Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.
18.
Gema López-Torrejón, Stefan Burén, Marcel Veldhuizen, Luis M. Rubio
Microb Biotechnol., vol. 14, iss. 3, pp. 1073-1083, 2021.
@article{nokey,
title = {Biosynthesis of cofactor‐activatable iron‐only nitrogenase in \emph{Saccharomyces cerevisiae}},
author = {Gema López-Torrejón and Stefan Burén and Marcel Veldhuizen and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085987/pdf/MBT2-14-1073.pdf},
doi = {10.1111/1751-7915.13758},
year = {2021},
date = {2021-05-25},
urldate = {2021-05-25},
journal = {Microb Biotechnol.},
volume = {14},
issue = {3},
pages = {1073-1083},
abstract = {Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe‐only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe‐only nitrogenase component proteins in the low‐oxygen mitochondria matrix of \emph{S. cerevisiae}. As‐isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo‐AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo‐AnfDK could be activated for acetylene reduction by the simple addition of FeMo‐co \emph{in vitro}, indicating preexistence of the P‐clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe‐only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.
Fe‐only nitrogenase components were expressed and matured in \emph{S. cerevisiae} mitochondrial. The electron donor component was active as isolated and the catalytic component was ready for activation by cofactor insertion.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe‐only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe‐only nitrogenase component proteins in the low‐oxygen mitochondria matrix of S. cerevisiae. As‐isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo‐AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo‐AnfDK could be activated for acetylene reduction by the simple addition of FeMo‐co in vitro, indicating preexistence of the P‐clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe‐only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.
Fe‐only nitrogenase components were expressed and matured in S. cerevisiae mitochondrial. The electron donor component was active as isolated and the catalytic component was ready for activation by cofactor insertion.
17.
Xi Jiang, Lucía Payá-Tormo, Diana Coroian, Inés García-Rubio, Rocío Castellanos-Rueda, Álvaro Eseverri, Gema López-Torrejón, Stefan Burén (corresponding author), Luis M. Rubio
Commun Biol., vol. 4, iss. 1, pp. 4, 2021.
@article{nokey,
title = {Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N_{2}-fixation in plants},
author = {Xi Jiang and Lucía Payá-Tormo and Diana Coroian and Inés García-Rubio and Rocío Castellanos-Rueda and Álvaro Eseverri and Gema López-Torrejón and Stefan Burén (corresponding author) and Luis M. Rubio},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782807/pdf/42003_2020_Article_1536.pdf},
doi = {10.1038/s42003-020-01536-6},
year = {2021},
date = {2021-01-04},
urldate = {2021-01-04},
journal = {Commun Biol.},
volume = {4},
issue = {1},
pages = {4},
abstract = {Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase \emph{nifH} sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. \emph{Hydrogenobacter thermophilus} (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N_{2} fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. \emph{H. thermophilus} NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe_{4}S_{4}] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.
16.
Zhi-Yong Yang, Emilio Jimenez-Vicente, Hayden Kallas, Dmitriy A. Lukoyanov, Hao Yang, Julia S. Martin del Campo, Dennis R. Dean, Brian M. Hoffman, Lance C. Seefeldt
The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase
Chem. Sci., vol. 12, iss. 20, pp. 6913-6922, 2021.
@article{nokey,
title = {The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase},
author = {Zhi-Yong Yang and Emilio Jimenez-Vicente and Hayden Kallas and Dmitriy A. Lukoyanov and Hao Yang and Julia S. Martin del Campo and Dennis R. Dean and Brian M. Hoffman and Lance C. Seefeldt},
url = {https://pubs.rsc.org/en/content/articlepdf/2021/sc/d0sc06561g},
doi = {10.1039/D0SC06561G},
year = {2021},
date = {2021-03-29},
urldate = {2021-03-29},
journal = {Chem. Sci.},
volume = {12},
issue = {20},
pages = {6913-6922},
publisher = {The Royal Society of Chemistry},
abstract = {The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad \emph{S} = 3/2 EPR signal (Kramers state) having \emph{g} values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V^{3+}, 3Fe^{3+}, 4Fe^{2+}]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, \emph{S} = 0, or non-Kramers, integer-spin (\emph{S} = 1, 2 \emph{etc.}). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic \emph{S} = 1/2 EPR signal from such a reduced state is observed, with \emph{g} = [2.18, 2.12, 2.09] and showing well-defined ^{51}V (\emph{I} = 7/2) hyperfine splitting, \emph{a}_{iso} = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: \emph{S} = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V^{3+}, 4Fe^{3+}, 3Fe^{2+}]. Our findings suggest that the V^{3+} does not change valency throughout the catalytic cycle.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V<small>3+</small>, 3Fe<small>3+</small>, 4Fe<small>2+</small>]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined <small>51</small>V (I = 7/2) hyperfine splitting, a<small>iso</small> = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V<small>3+</small>, 4Fe<small>3+</small>, 3Fe<small>2+</small>]. Our findings suggest that the V<small>3+</small> does not change valency throughout the catalytic cycle.
2020
15.
Álvaro Eseverri, Can Baysal, Vicente Medina, Teresa Capell, Paul Christou, Luis M. Rubio, Elena Caro
Front. Plant Sci., vol. 11, 2020, ISSN: 1664-462X.
@article{nokey,
title = {Transit peptides from photosynthesis-related proteins mediate import of a marker protein into different plastid types and within different species},
author = {Álvaro Eseverri and Can Baysal and Vicente Medina and Teresa Capell and Paul Christou and Luis M. Rubio and Elena Caro},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2020.560701/pdf},
doi = {10.3389/fpls.2020.560701},
issn = {1664-462X},
year = {2020},
date = {2020-09-25},
urldate = {2020-09-25},
journal = {Front. Plant Sci.},
volume = {11},
abstract = {Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four \emph{Arabidopsis thaliana} proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In \emph{A. thaliana}, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.
14.
Álvaro Eseverri, Gema López-Torrejón, Xi Jiang, Stefan Burén, Luis M. Rubio, Elena Caro
Plant Biotechnol J., vol. 18, no. 9, pp. 1882-1896, 2020.
@article{nokey,
title = {Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells},
author = {Álvaro Eseverri and Gema López-Torrejón and Xi Jiang and Stefan Burén and Luis M. Rubio and Elena Caro},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.13347},
doi = {10.1111/pbi.13347},
year = {2020},
date = {2020-01-27},
journal = {Plant Biotechnol J.},
volume = {18},
number = {9},
pages = {1882-1896},
abstract = {The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. \emph{Azotobacter vinelandii} nitrogen fixation genes, \emph{nifH}, \emph{M}, \emph{U} and \emph{S,} were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of \emph{n}\emph{ifU} and \emph{n}\emph{ifS} co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.
13.
Stefan Burén, Emilio Jiménez-Vicente, Carlos Echavarri-Erasun, Luis M. Rubio
Biosynthesis of nitrogenase cofactors
Chem. Rev., vol. 120, iss. 12, pp. 4921–4968, 2020, ISSN: 0009-2665.
@article{nokey,
title = {Biosynthesis of nitrogenase cofactors},
author = {Stefan Burén and Emilio Jiménez-Vicente and Carlos Echavarri-Erasun and Luis M. Rubio},
url = {https://doi.org/10.1021/acs.chemrev.9b00489},
doi = {10.1021/acs.chemrev.9b00489},
issn = {0009-2665},
year = {2020},
date = {2020-06-24},
urldate = {2020-06-24},
journal = {Chem. Rev.},
volume = {120},
issue = {12},
pages = {4921–4968},
publisher = {American Chemical Society},
abstract = {Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo-\emph{R}-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both \emph{in situ}, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo-R-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both in situ, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.
2019
12.
Stefan Burén, Katelin Pratt, Xi Jiang, Yisong Guo, Emilio Jimenez-Vicente, Carlos Echavarri-Erasun, Dennis R. Dean, Ishtiaq Saaem, D. Benjamin Gordon, Christopher A. Voigt, Luis M. Rubio
PNAS, vol. 116, no. 50, pp. 25078-25086, 2019.
@article{nokey,
title = {Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in \emph{Saccharomyces cerevisiae}},
author = {Stefan Burén and Katelin Pratt and Xi Jiang and Yisong Guo and Emilio Jimenez-Vicente and Carlos Echavarri-Erasun and Dennis R. Dean and Ishtiaq Saaem and D. Benjamin Gordon and Christopher A. Voigt and Luis M. Rubio},
url = {https://www.pnas.org/doi/abs/10.1073/pnas.1904903116},
doi = {10.1073/pnas.1904903116},
year = {2019},
date = {2019-11-25},
journal = {PNAS},
volume = {116},
number = {50},
pages = {25078-25086},
abstract = {The radical \textit{S}-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured \textit{Saccharomyces cerevisiae}. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted \textit{nif} genes (\emph{nifUSXB} and \emph{fdxN}) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including \emph{nifUSX} and \emph{fdxN}, and a library consisting of 28 different \emph{nifB} genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from \emph{Azotobacter vinelandii} with NifB from the archaea \emph{Methanocaldococcus infernus} or \emph{Methanothermobacter thermautotrophicus} yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured \emph{S. cerevisiae} coexpressing \emph{M. thermautotrophicus} NifB with \emph{A. vinelandii} NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.
11.
Can Baysal, Ana Pérez-González, Álvaro Eseverri, Xi Jiang, Vicente Medina, Elena Caro, Luis M. Rubio, Paul Christou, Changfu Zhu
Transgenic Res., vol. 29, pp. 37-52, 2019.
@article{nokey,
title = {Recognition motifs rather than phylogenetic origin influence the ability of targeting peptides to import nuclear-encoded recombinant proteins into rice mitochondria},
author = {Can Baysal and Ana Pérez-González and Álvaro Eseverri and Xi Jiang and Vicente Medina and Elena Caro and Luis M. Rubio and Paul Christou and Changfu Zhu},
url = {https://link.springer.com/article/10.1007/s11248-019-00176-9},
doi = {10.1007/s11248-019-00176-9},
year = {2019},
date = {2019-10-10},
journal = {Transgenic Res.},
volume = {29},
pages = {37-52},
abstract = {Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (\emph{Oryza sativa}). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (\emph{Saccharomyces cerevisiae}), SU9 (\emph{Neurospora crassa}), pFA (\emph{Arabidopsis thaliana}) and OsSCSb (\emph{Oryza sativa}) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (\emph{Nicotiana plumbaginifolia}) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.
10.
Mónica Navarro-Rodríguez, José María Buesa, Luis M. Rubio
Front. Microbiol., vol. 10, pp. 579, 2019.
@article{nokey,
title = {Genetic and biochemical analysis of the \emph{Azotobacter vinelandii} molybdenum storage protein},
author = {Mónica Navarro-Rodríguez and José María Buesa and Luis M. Rubio},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2019.00579/full},
doi = {10.3389/fmicb.2019.00579},
year = {2019},
date = {2019-03-21},
urldate = {2019-03-21},
journal = {Front. Microbiol.},
volume = {10},
pages = {579},
abstract = {The N_{2} fixing bacterium \emph{Azotobacter vinelandii} carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The \emph{in vitro} FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an \emph{A. vinelandii} strain lacking the MoSto subunit genes (Δ\emph{mosAB}) was analyzed. Consistent with its role as storage protein, the Δ\emph{mosAB} strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased \emph{in vivo} nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The Δ\emph{mosAB} strain was found to readily derepress \emph{vnfDGK} upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The Δ\emph{mosAB} mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δ\emph{vnf} Δ\emph{anf}) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the Δ\emph{mosAB} and \emph{mosAB}^{+} strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to \emph{A. vinelandii} in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The N2 fixing bacterium Azotobacter vinelandii carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron–molybdenum cofactor (FeMo-co). The in vitro FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation. The phenotype of an A. vinelandii strain lacking the MoSto subunit genes (ΔmosAB) was analyzed. Consistent with its role as storage protein, the ΔmosAB strain showed severe impairment to accumulate intracellular Mo and lower resilience than wild type to Mo starvation as demonstrated by decreased in vivo nitrogenase activity and competitive growth index. In addition, it was more sensitive than the wild type to diazotrophic growth inhibition by W. The ΔmosAB strain was found to readily derepress vnfDGK upon Mo step down, in contrast to the wild type that derepressed Vnf proteins only after prolonged Mo starvation. The ΔmosAB mutation was then introduced in a strain lacking V and Fe-only nitrogenase structural genes (Δvnf Δanf) to investigate possible compensations from these alternative systems. When grown in Mo-depleted medium, the ΔmosAB and mosAB+ strains showed low but similar nitrogenase activities regardless of the presence of Vnf proteins. This study highlights the selective advantage that MoSto confers to A. vinelandii in situations of metal limitation as those found in many soil ecosystems. Such a favorable trait should be included in the gene complement of future nitrogen fixing plants.
9.
Emilio Jimenez-Vicente, Zhi-Yong Yang, Julia S. Martin del Campo, Valerie L. Cash, Lance C. Seefeldt, Dennis R. Dean
J. Biol. Chem., vol. 294, iss. 16, pp. 6204-6213, 2019.
@article{nokey,
title = {The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters},
author = {Emilio Jimenez-Vicente and Zhi-Yong Yang and Julia S. Martin del Campo and Valerie L. Cash and Lance C. Seefeldt and Dennis R. Dean},
doi = {10.1074/jbc.RA119.007905},
year = {2019},
date = {2019-04-19},
urldate = {2019-04-19},
journal = {J. Biol. Chem.},
volume = {294},
issue = {16},
pages = {6204-6213},
abstract = {The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α_{2}β_{2} heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N_{2} reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N_{2}-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as \emph{in vivo}, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe \emph{Azotobacter vinelandii}. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature \emph{in vitro} without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α2β2 heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein’s catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity–based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.
2018
8.
Stefan Burén, Gema López-Torrejón, Luis M. Rubio
Extreme bioengineering to meet the nitrogen challenge
PNAS, vol. 115, no. 36, pp. 8849-8851, 2018.
@article{nokey,
title = {Extreme bioengineering to meet the nitrogen challenge},
author = {Stefan Burén and Gema López-Torrejón and Luis M. Rubio},
url = {https://www.pnas.org/doi/abs/10.1073/pnas.1812247115},
doi = {10.1073/pnas.1812247115},
year = {2018},
date = {2018-08-16},
journal = {PNAS},
volume = {115},
number = {36},
pages = {8849-8851},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
7.
Emilio Jimenez-Vicente, Zhi-Yong Yang, W. Keith Ray, Carlos Echavarri-Erasun, Valerie L. Cash, Luis M. Rubio, Lance C. Seefeldt, Dennis R. Dean
J. Biol. Chem., vol. 293, iss. 25, pp. 9812-9823, 2018, ISSN: 0021-9258.
@article{nokey,
title = {Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation},
author = {Emilio Jimenez-Vicente and Zhi-Yong Yang and W. Keith Ray and Carlos Echavarri-Erasun and Valerie L. Cash and Luis M. Rubio and Lance C. Seefeldt and Dennis R. Dean},
url = {https://www.sciencedirect.com/science/article/pii/S0021925820399658},
doi = {10.1074/jbc.RA118.002994},
issn = {0021-9258},
year = {2018},
date = {2018-06-12},
urldate = {2018-06-12},
journal = {J. Biol. Chem.},
volume = {293},
issue = {25},
pages = {9812-9823},
abstract = {Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium \emph{Azotobacter vinelandii}. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an \emph{A. vinelandii} strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
2017
6.
Stefan Burén, Luis M. Rubio
State of the art in eukaryotic nitrogenase engineering
FEMS Microbiol. Lett., vol. 365, iss. 2, 2017, ISSN: 0378-1097.
@article{nokey,
title = {State of the art in eukaryotic nitrogenase engineering},
author = {Stefan Burén and Luis M. Rubio},
url = {https://doi.org/10.1093/femsle/fnx274},
doi = {10.1093/femsle/fnx274},
issn = {0378-1097},
year = {2017},
date = {2017-12-12},
journal = {FEMS Microbiol. Lett.},
volume = {365},
issue = {2},
abstract = {Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.
5.
Simon Arragain, Emilio Jiménez-Vicente, Alessandro A. Scandurra, Stefan Burén, Luis M. Rubio, Carlos Echavarri-Erasun
Diversity and functional analysis of the FeMo-cofactor maturase NifB
Front. Plant Sci., vol. 8, 2017, ISSN: 1664-462X.
@article{nokey,
title = {Diversity and functional analysis of the FeMo-cofactor maturase NifB},
author = {Simon Arragain and Emilio Jiménez-Vicente and Alessandro A. Scandurra and Stefan Burén and Luis M. Rubio and Carlos Echavarri-Erasun},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2017.01947/pdf},
doi = {10.3389/fpls.2017.01947},
issn = {1664-462X},
year = {2017},
date = {2017-11-14},
urldate = {2017-11-14},
journal = {Front. Plant Sci.},
volume = {8},
abstract = {One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile \emph{Chlorobium tepidum} (two-domain architecture), the hyperthermophile \emph{Methanocaldococcus infernus} (single-domain architecture) and the mesophile \emph{Klebsiella oxytoca} (two-domain architecture), showing \emph{in silico} characterization of their nitrogen fixation (\emph{nif}) gene clusters, conserved NifB motifs, and functionality. \emph{C. tepidum} and \emph{M. infernus} NifB were able to complement an \emph{Azotobacter vinelandii} (Δ\emph{nifB}) mutant restoring the Nif^{+} phenotype and thus demonstrating their functionality \emph{in vivo}. In addition, purified \emph{C. tepidum} NifB exhibited activity in the \emph{in vitro} NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain \emph{K. oxytoca} NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher \emph{in vivo} nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
4.
Ana Pérez-González, Ryan Kniewel, Marcel Veldhuizen, Hemant K. Verma, Mónica Navarro-Rodríguez, Luis M. Rubio, Elena Caro
BMC Biotechnol., vol. 17, no. 80, 2017, ISSN: 1472-6750.
@article{nokey,
title = {Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria},
author = {Ana Pérez-González and Ryan Kniewel and Marcel Veldhuizen and Hemant K. Verma and Mónica Navarro-Rodríguez and Luis M. Rubio and Elena Caro},
url = {https://doi.org/10.1186/s12896-017-0393-y},
doi = {10.1186/s12896-017-0393-y},
issn = {1472-6750},
year = {2017},
date = {2017-11-13},
urldate = {2017-11-13},
journal = {BMC Biotechnol.},
volume = {17},
number = {80},
abstract = {\textbf{Background}
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
\textbf{Results}
Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within \emph{Saccharomyces cerevisiae}. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (\emph{nif}) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, \emph{nifU}, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.
\textbf{Conclusions}
We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Background
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
Results
Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.
Conclusions
We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.
3.
Stefan Burén, Xi Jiang, Gema López-Torrejón, Carlos Echavarri-Erasun, Luis M. Rubio
Front. Plant Sci., vol. 8, 2017, ISSN: 1664-462X.
@article{nokey,
title = {Purification and \emph{in vitro} activity of mitochondria targeted nitrogenase cofactor maturase NifB},
author = {Stefan Burén and Xi Jiang and Gema López-Torrejón and Carlos Echavarri-Erasun and Luis M. Rubio},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2017.01567/full},
doi = {10.3389/fpls.2017.01567},
issn = {1664-462X},
year = {2017},
date = {2017-09-12},
urldate = {2017-09-12},
journal = {Front. Plant Sci.},
volume = {8},
abstract = {Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O_{2}-sensitive \emph{S}-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium \emph{Azotobacter vinelandii} and the thermophilic methanogen \emph{Methanocaldococcus infernus} in \emph{Saccharomyces cerevisiae} and in \emph{Nicotiana benthamiana}. NifB proteins were targeted to the mitochondria, where O_{2} consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the \emph{A. vinelandii} version appeared to form aggregates. Similarly, NifB from \emph{M. infernus} was expressed at higher levels in leaves of \emph{Nicotiana benthamiana} and accumulated as a soluble protein while \emph{A. vinelandii} NifB was mainly associated with the non-soluble cell fraction. Soluble \emph{M. infernus} NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the \emph{in vitro} FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening \emph{nif} genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.
2.
Stefan Burén, Eric M. Young, Elizabeth A. Sweeny, Gema Lopez-Torrejón, Marcel Veldhuizen, Christopher A. Voigt, Luis M. Rubio
ACS Synth. Biol., vol. 6, iss. 6, pp. 1043–1055, 2017.
@article{nokey,
title = {Formation of nitrogenase NifDK tetramers in the mitochondria of \emph{Saccharomyces cerevisiae}},
author = {Stefan Burén and Eric M. Young and Elizabeth A. Sweeny and Gema Lopez-Torrejón and Marcel Veldhuizen and Christopher A. Voigt and Luis M. Rubio},
url = {https://doi.org/10.1021/acssynbio.6b00371},
doi = {10.1021/acssynbio.6b00371},
year = {2017},
date = {2017-02-21},
urldate = {2017-02-21},
journal = {ACS Synth. Biol.},
volume = {6},
issue = {6},
pages = {1043–1055},
abstract = {Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N_{2} fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O_{2} consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of \emph{Saccharomyces cerevisiae} in which transcriptional units comprising nine \emph{Azotobacter vinelandii nif} genes (\emph{nifHDKUSMBEN}) were integrated into the genome. Two combinatorial libraries of \emph{nif} gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of \emph{nif} gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and \emph{nif} expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust \emph{nif} gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.
2016
1.
Gema López-Torrejón, Emilio Jiménez-Vicente, José María Buesa, Jose A. Hernandez, Hemant K. Verma, Luis M. Rubio
Nat. Commun., vol. 7, iss. 1, pp. 11426, 2016.
@article{nokey,
title = {Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast},
author = {Gema López-Torrejón and Emilio Jiménez-Vicente and José María Buesa and Jose A. Hernandez and Hemant K. Verma and Luis M. Rubio},
url = {https://doi.org/10.1038/ncomms11426},
doi = {10.1038/ncomms11426},
year = {2016},
date = {2016-04-29},
journal = {Nat. Commun.},
volume = {7},
issue = {1},
pages = {11426},
abstract = {The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct \emph{nif} gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N_{2}-fixing cereals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.
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