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Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

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 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.

SUBMITTER: He W

PROVIDER: S-EPMC9486962 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

He Wenshu W Burén Stefan S Baysal Can C Jiang Xi X Capell Teresa T Christou Paul P Rubio Luis M LM

ACS synthetic biology 20220823 9

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 ...[more]

PMID: 35998307

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Project description: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. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.

Dataset Information

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

Publications

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

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