Carlos Erasun

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

Biosynthesis of Nitrogenase Cofactors

Chemical Reviews

Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. Th... more 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. CONTENTS 7.1. NifU and NifS 7.2. Fe Protein Is Required for P-Cluster Formation 7.3. NifZ Is Involved in P-Cluster Formation 7.3.1. Model 1: NifZ Is Only Required for Maturation of Second P-Cluster in Each Apo-MoFe Protein Molecule 7.3.2. Model 2: NifZ Is Involved in Maturation of Both P-Clusters 8. FeMo-co: Description of the Cofactor and Methods to Measure Its Biosynthesis 8.1. Discovery and Isolation of FeMo-co 8.2. In Vitro Systems for FeMo-Cofactor Synthesis and Insertion 9. Model for FeMo-co Biosynthesis 10. Biosynthesis of FeMo-co Fe-S Core: Roles of NifU, NifS, NifB, and FdxN 10.1. NifS and NifU Assembly of Precursor [Fe-S] Clusters for FeMo-co 10.2. NifB and NifB-co 10.2.1. Information from nif B Mutagenesis 10.2.2. Identification and Isolation of NifB-co, the Product of NifB Activity 10.2.3. Interstitial Atom of FeMo-co Is Present at NifB-co

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

Proceedings of the National Academy of Sciences

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in n... more 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 ...

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Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB

Frontiers in plant science, 2017

One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB acti... more 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 (two-domain architecture), the hyperthermophile (single-domain architecture) and the mesophile (two-domain architecture), showing characterization of their nitrogen fixation () gene clusters, conserved NifB motifs, and functionality. and NifB were able to complement an (Δ) mutant restoring the Nif phenotype and thus demonst...

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

The Journal of biological chemistry, Jan 3, 2018

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitr... more Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-Chomocitrate] 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 We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an 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. ...

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

Frontiers in Plant Science

Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extre... more Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O 2-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 codonoptimized 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 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 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.

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

Angewandte Chemie International Edition, 2016

The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. F... more 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.

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

Chemical communications (Cambridge, England), Jan 27, 2016

Mo and Fe K-edge EXAFS analysis of NifQ shows the presence of a [MoFe3S4] cluster and a second in... more 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.

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

Journal of the American Chemical Society, Jun 22, 2016

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse tw... more 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.

Regulation of nif Gene Expression in Azotobacter vinelandii

deBruijn/Biological Nitrogen Fixation, 2015

Fungal carotenoids

Applied Mycology and Biotechnology, 2002

... Applied Mycology and Biotechnology Volume 2. Agriculture and Food Production 45 2002 Publishe... more

Yeast Biotechnology

The Yeasts, 2011

Expression and purification of NifB proteins from aerobic and anaerobic sources

Methods in molecular biology (Clifton, N.J.), 2014

NifB is the key protein in the biosynthesis of nitrogenase iron-molybdenum cofactor. Due to its e... more 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.

Purification of O2-Sensitive Metalloproteins

Methods in Molecular Biology, 2014

The most dependable factor to perform successful biochemical experiments in an O2-free environmen... more 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.

Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

Journal of Bacteriology, 2014

Nitrogen fixation is a tightly regulated trait. Switching from N 2 fixation-repressing conditions... more Nitrogen fixation is a tightly regulated trait. Switching from N 2 fixation-repressing conditions to the N 2 -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 structura...

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Yeast Genome 10th Anniversary, AcadÃ©mie Royale des Sciences, des Letters et des Beaux-Arts de Belgique in Brussels (Belgium), 7â8 September 2006

FEMS Yeast Research, 2006

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Stimulation of astaxanthin formation in the yeast by the fungus

FEMS Yeast Research, 2004

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous marked... more 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 (L-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 L-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.

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