Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae.

Biological nitrogen fixation, the conversion of inert N2 to metabolically tractable NH3, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo-R-homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae, represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae, targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii. However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae. Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo-R-homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae. NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae. FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.

Iron Homeostasis in Azotobacter vinelandii.

Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while preventing toxicity. Iron uptake and exchange networks must adapt to the different environmental conditions, particularly those that require the biosynthesis of multiple iron proteins, such as nitrogen fixation. In this review, we outline the mechanisms that the model diazotrophic bacterium Azotobacter vinelandii uses to ensure iron nutrition and how it adapts Fe metabolism to diazotrophic growth.

Forging a symbiosis: transition metal delivery in symbiotic nitrogen fixation.

Symbiotic nitrogen fixation carried out by the interaction between legumes and rhizobia is the main source of nitrogen in natural ecosystems and in sustainable agriculture. For the symbiosis to be viable, nutrient exchange between the partners is essential. Transition metals are among the nutrients delivered to the nitrogen-fixing bacteria within the legume root nodule cells. These elements are used as cofactors for many of the enzymes controlling nodule development and function, including nitrogenase, the only known enzyme able to convert N2 into NH3 . In this review, we discuss the current knowledge on how iron, zinc, copper, and molybdenum reach the nodules, how they are delivered to nodule cells, and how they are transferred to nitrogen-fixing bacteria within.

Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana.

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.

Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme.

Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.

Biosynthesis of Nitrogenase Cofactors.

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.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae.

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.

Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation.

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.

Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB.

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.

Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB.

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.

EXAFS reveals two Mo environments in the nitrogenase iron-molybdenum cofactor biosynthetic protein NifQ.

Mo and Fe K-edge EXAFS analysis of NifQ shows the presence of a [MoFe3S4] cluster and a second independent Mo environment that includes Mo-O bonds and Mo-S bonds. Both environments are relevant to FeMo-co biosynthesis and may represent different stages of Mo biochemical transformations catalyzed by NifQ.

The Nitrogenase FeMo-Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms.

The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mössbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.

Electron Paramagnetic Resonance Characterization of Three Iron-Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus.

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.

Purification of O2-sensitive metalloproteins.

The most dependable factor to perform successful biochemical experiments in an O2-free environment is the experience required to set up an efficient laboratory, to properly manipulate samples, to anticipate potential O2-related problems, and to maintain the complex laboratory setup operative. There is a long list of O2-related issues that may ruin your experiments. We provide here a guide to minimize these risks.

Expression and purification of NifB proteins from aerobic and anaerobic sources.

NifB is the key protein in the biosynthesis of nitrogenase iron-molybdenum cofactor. Due to its extreme sensitivity to O2 and inherent protein instability, NifB proteins must be purified under strict anaerobic conditions by using affinity chromatography methods. We describe here the methods for NifB purification from cells of the strict aerobic nitrogen-fixing bacterium Azotobacter vinelandii, the facultative anaerobic nitrogen-fixing bacterium Klebsiella pneumoniae, and the facultative anaerobic non-nitrogen fixing bacterium Escherichia coli recombinantly expressing a nifB gene of thermophilic origin.

Kinetics of Nif gene expression in a nitrogen-fixing bacterium.

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.

Cophenetic correlation analysis as a strategy to select phylogenetically informative proteins: an example from the fungal kingdom.

BACKGROUND: The construction of robust and well resolved phylogenetic trees is important for our understanding of many, if not all biological processes, including speciation and origin of higher taxa, genome evolution, metabolic diversification, multicellularity, origin of life styles, pathogenicity and so on. Many older phylogenies were not well supported due to insufficient phylogenetic signal present in the single or few genes used in phylogenetic reconstructions. Importantly, single gene phylogenies were not always found to be congruent. The phylogenetic signal may, therefore, be increased by enlarging the number of genes included in phylogenetic studies. Unfortunately, concatenation of many genes does not take into consideration the evolutionary history of each individual gene. Here, we describe an approach to select informative phylogenetic proteins to be used in the Tree of Life (TOL) and barcoding projects by comparing the cophenetic correlation coefficients (CCC) among individual protein distance matrices of proteins, using the fungi as an example. The method demonstrated that the quality and number of concatenated proteins is important for a reliable estimation of TOL. Approximately 40-45 concatenated proteins seem needed to resolve fungal TOL. RESULTS: In total 4852 orthologous proteins (KOGs) were assigned among 33 fungal genomes from the Asco- and Basidiomycota and 70 of these represented single copy proteins. The individual protein distance matrices based on 531 concatenated proteins that has been used for phylogeny reconstruction before 14 were compared one with another in order to select those with the highest CCC, which then was used as a reference. This reference distance matrix was compared with those of the 70 single copy proteins selected and their CCC values were calculated. Sixty four KOGs showed a CCC above 0.50 and these were further considered for their phylogenetic potential. Proteins belonging to the cellular processes and signaling KOG category seem more informative than those belonging to the other three categories: information storage and processing; metabolism; and the poorly characterized category. After concatenation of 40 proteins the topology of the phylogenetic tree remained stable, but after concatenation of 60 or more proteins the bootstrap support values of some branches decreased, most likely due to the inclusion of proteins with lowers CCC values. The selection of protein sequences to be used in various TOL projects remains a critical and important process. The method described in this paper will contribute to a more objective selection of phylogenetically informative protein sequences. CONCLUSION: This study provides candidate protein sequences to be considered as phylogenetic markers in different branches of fungal TOL. The selection procedure described here will be useful to select informative protein sequences to resolve branches of TOL that contain few or no species with completely sequenced genomes. The robust phylogenetic trees resulting from this method may contribute to our understanding of organismal diversification processes. The method proposed can be extended easily to other branches of TOL.

Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum.

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.