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Sample records for nitrogenase femo cofactor

  1. FeMo cofactor of nitrogenase: energetics and local interactions in the protein environment.

    PubMed

    Lovell, Timothy; Li, Jian; Case, David A; Noodleman, Louis

    2002-09-01

    A combined broken-symmetry density functional and continuum electrostatics approach has been applied to the iron-molybdenum center (FeMoco) of nitrogenase to evaluate the energetic effects of the local amino acid environment for several spin alignments of FeMoco. The protein environment preferentially stabilizes certain spin coupling patterns. The lowest energy spin alignment pattern in the protein displays calculated properties that match the experimental data better than any of the alternative possibilities. The total interaction energy of the protein with FeMoco has been evaluated and the contribution of each amino acid residue has been broken down into sidechain and backbone components. Arginine, lysine, aspartate and glutamate sidechains exert the largest electrostatic influence on FeMoco; specific residues are highlighted and their interaction with FeMoco discussed in the context of the available X-ray data from Azotobacter vinelandii (Av). Observed data for the M(N)(resting state)-->M(OX)(one-electron oxidized state) and M(N)-->M(R)(one-electron reduced state) or M(I)(alternative one-electron reduced state) redox couples are compared with those calculated for Av. The calculated redox potentials are fairly insensitive to the spin state of the oxidized or reduced states and the predicted qualitative trend of a more negative redox potential for the more reduced M(N)-->M(R) or M(I) couple is in accord with the available redox data. These calculations represent a first step towards the development of a microscopic model of electron and proton transfer events at the nitrogenase active site.

  2. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    DOE PAGES

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; ...

    2014-12-04

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of thesemore » remarkable metal cofactors.« less

  3. The Fe-V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom.

    PubMed

    Rees, Julian A; Bjornsson, Ragnar; Schlesier, Julia; Sippel, Daniel; Einsle, Oliver; DeBeer, Serena

    2015-11-02

    The first direct evidence is provided for the presence of an interstitial carbide in the Fe-V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe-Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme.

  4. Radical S-Adenosyl-L-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

    SciTech Connect

    Byer, Amanda S.; Shepard, Eric M.; Peters, John W.; Broderick, Joan B.

    2014-12-04

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. Furthermore, the FeMo cofactor of nitrogenase has a MoFe7S9 cluster with a central carbon, whereas the H-cluster of [FeFe]-hydrogenase contains a 2Fe subcluster coordinated by cyanide and CO ligands as well as dithiomethylamine; the [Fe]-hydrogenase cofactor has CO and guanylylpyridinol ligands at a mononuclear iron site. Intriguingly, radical S-adenosyl-L-methionine enzymes are vital for the assembly of all three of these diverse cofactors. Here, in this minireview, we present and discuss the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors.

  5. Protocols for cofactor isolation of nitrogenase.

    PubMed

    Fay, Aaron W; Lee, Chi-Chung; Wiig, Jared A; Hu, Yilin; Ribbe, Markus W

    2011-01-01

    The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein has remained a focal point in the field of bioinorganic chemistry for decades. This unique metal cluster has long been regarded as the actual site of dinitrogen reduction, and it is structurally complex and chemically unprecedented. A detailed characterization of the isolated FeMoco is crucial for elucidating the physiochemical properties of this biologically important cofactor. Such a study requires an effective technique to extract FeMoco intact, and in high yield, from the MoFe protein. A method involving the acid treatment of the MoFe protein and the subsequent extraction of FeMoco into an organic solvent was developed over 30 years ago and has been improved upon ever since. FeMoco isolated by this strategy is catalytically active and spectrally interesting, which provides a useful platform for future structure-function analyses of this unique cofactor. A general working protocol for FeMoco isolation is described in this chapter, along with some of the major modifications reported in the past years.

  6. Structural studies of the molybdenum site in the MoFe protein and it FeMo cofactor by EXAFS

    SciTech Connect

    Conradson, S.D.; Burgess, B.K.; Newton, W.E.; Mortenson, L.E.; Hodgson, K.O.

    1987-11-25

    Nitrogenase is a complex bacterial enzyme system that is responsible for the conversion of atmospheric N/sub 2/ to ammonia. The structure and function of molybdenum in the MoFe protein of this system has been the subject of a number of investigations, including the use of X-ray absorption spectroscopy. This paper reports the results of the authors recent studies on several states of the MoFe protein and its FeMo cofactor (which is extruded by treatment with N-methylformamide). Mo K-edge (XANES) and extended fine structure (EXAFS) spectra have been recorded to high energies above the absorption edge with excellent signal-to-nose on the semireduced form of the MoFe protein from both Clostridium pasteurianum and Azotobacter vinelandii and on the as isolated FeMo-co and FeMo-co treated with benzenethiol and with benzeneselenol. In all of the states studied, EXAFS results reveal that the Mo is in an environment that contains two or three oxygen (or nitrogen) atoms at 2.10-2.12 A, three to five S atoms at 2.37 A, and three to four Fe atoms at 2.68-2.70 A. The numbers of these ligands change upon removal of the cofactor from the protein as discussed in the paper. For FeMo-co, comparisons also show that thil/selenol is not binding directly to the Mo site. The results of these EXAFS (and their XANES published earlier) definitely show the presence of several low-Z ligands and are not compatible with a tetrahedral arrangement of only nearest S neighbors at the Mo site.

  7. Insights into hydrocarbon formation by nitrogenase cofactor homologs.

    PubMed

    Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W

    2015-04-14

    The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN(-) to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN(-), which could be explained by the presence of a "free" Fe atom that is "unmasked" by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C-C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN(-) reduction without complications originating from the heterometal and homocitrate components. Nitrogenase is a metalloenzyme that is highly complex in structure and uniquely versatile in function. It catalyzes two reactions that parallel two important industrial processes: the reduction of nitrogen to ammonia, which parallels the Haber-Bosch process in ammonia production, and the reduction of carbon monoxide to hydrocarbons, which parallels the Fischer-Tropsch process in fuel production. Thus, the significance of nitrogenase can be appreciated from the perspective of the useful products it generates: (i) ammonia, the "fixed" nitrogen that is essential for the existence of the entire human population; and (ii) hydrocarbons, the "recycled" carbon fuel that could be used to directly address the worldwide energy shortage. This article provides initial insights into the catalytic characteristics of various nitrogenase cofactors in hydrocarbon formation. The reported assay system provides a useful tool for mechanistic

  8. Insights into Hydrocarbon Formation by Nitrogenase Cofactor Homologs

    PubMed Central

    Lee, Chi Chung; Hu, Yilin

    2015-01-01

    ABSTRACT The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN− to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN−, which could be explained by the presence of a “free” Fe atom that is “unmasked” by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C−C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN− reduction without complications originating from the heterometal and homocitrate components. PMID:25873377

  9. A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and preextraction treatments.

    PubMed

    McLean, P A; Wink, D A; Chapman, S K; Hickman, A B; McKillop, D M; Orme-Johnson, W H

    1989-11-28

    A convenient and rapid method of obtaining the cofactor of nitrogenase (FeMoco) with a low and apparently limiting Fe/Mo ratio has been developed. FeMoco can be extracted from the MoFe protein bound to DEAE-cellulose. The cofactor is eluted in either N-methylformamide (NMF), N,N-dimethylformamide (DMF), or mixtures of these solvents by use of salts such as Et4NBr,Bu4NBr,Ph4PCl, and Ph4AsCl. The method is simple, is rapid (45 min), yields concentrated cofactor, and, unlike the original method [Shah, V. K., & Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253] which requires anaerobic centrifugation, is easily scaled up. Furthermore, it gives yields of cofactor in excess of 70%. Its disadvantages are a high Fe:Mo ratio when DMF is the extracting solvent and a high salt concentration in the resultant FeMoco solution. These disadvantages are easily overcome by removing excess Fe by pretreating the cofactor with bipyridyl while still on the column. This gives Fe:Mo ratios of (6 +/- 1):1 (11 trials) with specific activities ranging from 170 to 220 nmol of C2H4/[min.(nmol of Mo)]. Chromatography on Sephadex LH-20 removes ca. 99% of the excess salt. The adsorption of MoFe protein to DEAE-cellulose seems to facilitate denaturation by organic solvents so that pretreatment of the protein with acid, used in earlier methods, is unnecessary. There is an apparent dependence on the charge density of the anion employed for elution of FeMoco bound to DEAE-cellulose, such that Cl- greater than Br- much greater than I-, PF6- is the order of effectiveness of the Bu4N+ salts of these anions.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions.

    PubMed

    Noumsi, Christelle Jouogo; Pourhassan, Nina; Darnajoux, Romain; Deicke, Michael; Wichard, Thomas; Burrus, Vincent; Bellenger, Jean-Philippe

    2016-02-01

    Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems. Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii. In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A.; Einsle, Oliver; Howard, James B.; Rees, Douglas C.

    2014-01-01

    The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO) inhibited nitrogenase MoFe-protein at 1.50 Å resolution, revealing a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The μ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 Å resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase. PMID:25258081

  12. Catalytic reduction of CN−, CO and CO2 by nitrogenase cofactors in lanthanide-driven reactions**

    PubMed Central

    Lee, Chi Chung

    2014-01-01

    Nitrogenase cofactors can be extracted into an organic solvent and added in an adenosine triphosphate (ATP)-free, organic solvent-based reaction medium to catalyze the reduction of cyanide (CN−), carbon monoxide (CO) and carbon dioxide (CO2) when samarium (II) iodide (SmI2) and 2,6-lutidinium triflate (Lut-H) are supplied as a reductant and a proton source, respectively. Driven by SmI2, the cofactors not only catalytically reduce CN− or CO to C1-C4 hydrocarbons, but also catalytically reduce CO2 to CO and C1-C3 hydrocarbons. The observation of C-C coupling from CO2 reveals a unique, Fischer-Tropsch-like reaction with an atypical carbonaceous substrate; whereas the achievement of catalytic turnover of CN−, CO and CO2 by isolated cofactors suggests the possibility to develop nitrogenase-based electrocatalysts for hydrocarbon production from these carbon-containing compounds. PMID:25420957

  13. The Fe–V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom

    PubMed Central

    Rees, Julian A; Bjornsson, Ragnar; Schlesier, Julia; Sippel, Daniel; Einsle, Oliver; DeBeer, Serena

    2015-01-01

    The first direct evidence is provided for the presence of an interstitial carbide in the Fe–V cofactor of Azotobacter vinelandii vanadium nitrogenase. As for our identification of the central carbide in the Fe–Mo cofactor, we employed Fe Kβ valence-to-core X-ray emission spectroscopy and density functional theory calculations, and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide, and thus an atomically homologous active site in vanadium nitrogenase, highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme. PMID:26376620

  14. Revisiting the Mössbauer Isomer Shifts of the FeMoco Cluster of Nitrogenase and the Cofactor Charge.

    PubMed

    Bjornsson, Ragnar; Neese, Frank; DeBeer, Serena

    2017-02-06

    Despite decades of research, the structure-activity relationship of nitrogenase is still not understood. Only recently was the full molecular structure of the FeMo cofactor (FeMoco) revealed, but the charge and metal oxidation states of FeMoco have been controversial. With the recent identification of the interstitial atom as a carbide and the more recent revised oxidation-state assignment of the molybdenum atom as Mo(III), here we revisit the Mössbauer properties of FeMoco. By a detailed error analysis of density functional theory-computed isomer shifts and computing isomer shifts relative to the P-cluster, we find that only the charge of [MoFe7S9C](1-) fits the experimental data. In view of the recent Mo(III) identification, the charge of [MoFe7S9C](1-) corresponds to a formal oxidation-state assignment of Mo(III)3Fe(II)4Fe(III), although due to spin delocalization, the physical oxidation state distribution might also be interpreted as Mo(III)1Fe(II)4Fe(2.5)2Fe(III), according to a localized orbital analysis of the MS = 3/2 broken symmetry solution. These results can be reconciled with the recent spatially resolved anomalous dispersion study by Einsle et al. that suggests the Mo(III)3Fe(II)4Fe(III) distribution, if some spin localization (either through interactions with the protein environment or through vibronic coupling) were to take place.

  15. The nitrogenase FeMo-cofactor and P-cluster pair: 2. 2 [Angstrom] resolution structures

    SciTech Connect

    Chan, M.K.; Jongsun Kim; Rees, D.C. )

    1993-05-07

    Structures recently proposed for the FeMo-cofactor and P-cluster pair of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii have been crystallographically verified at 2.2 angstrom resolution. Significantly, no hexacoordinate sulfur atoms are observed in either type of metal center. Consequently, the six bridged iron atoms in the FeMo-cofactor are trigonally coordinated by nonprotein ligands, although there may be some iron-iron bonding interactions that could provide a fourth coordination interaction for these sites. Two of the cluster sulfurs in the P-cluster pair are very close together ([approximately]2.1 angstroms), indicating that they form a disulfide bond. These findings indicate that a cavity exists in the interior of the FeMo-cofactor that could be involved in substrate binding and suggest that redox reactions at the P-cluster pair may be linked to transitions of two cluster-bound sulfurs between disulfide and sulfide oxidation states. 12 refs., 3 figs.

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

    PubMed

    Burén, Stefan; Jiang, Xi; López-Torrejón, Gema; Echavarri-Erasun, Carlos; Rubio, Luis M

    2017-01-01

    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.

  17. Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

    PubMed Central

    Fay, Aaron W.; Wiig, Jared A.; Lee, Chi Chung; Hu, Yilin

    2015-01-01

    Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally “truncated” homologs of NifB from Methanosarcina acetivorans (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifBMa and NifBMt are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifBMa and NifBMt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifBMa and NifBMt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5′-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifBMa and NifBMt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide. PMID:26627238

  18. Synthesis of the iron-molybdenum cofactor of nitrogenase is inhibited by a low-molecular-weight metabolite of Klebsiella pneumoniae.

    PubMed

    Downs, D M; Ludden, P W; Shah, V K

    1990-10-01

    The in vitro synthesis of the iron-molybdenum cofactor nitrogenase was inhibited by a low-molecular-weight factor. This inhibitory factor was present in the membrane extracts of wild-type and nif mutant strains of Klebsiella pneumoniae that were grown under conditions that either repressed or derepressed nitrogenase expression. In vitro, the inhibition was specific for the NifB protein. Addition of this factor to K. pneumoniae cells at various times during nif derepression decreased nitrogenase activity, presumably through inhibition of iron-molybdenum cofactor synthesis. The inhibitor was purified by solvent extraction and chromatography on DEAE-cellulose, silica gel, and aluminum oxide columns.

  19. Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition.

    PubMed

    Yang, S S; Pan, W H; Friesen, G D; Burgess, B K; Corbin, J L; Stiefel, E I; Newton, W E

    1982-07-25

    Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.

  20. Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein.

    PubMed

    Zhang, Li Mei; Morrison, Christine N; Kaiser, Jens T; Rees, Douglas C

    2015-02-01

    The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

  1. Model Calculations Suggest that the Central Carbon in the FeMo-Cofactor of Nitrogenase Becomes Protonated in the Process of Nitrogen Fixation.

    PubMed

    Siegbahn, Per E M

    2016-08-24

    Nitrogen activation by nitrogenase is one of the most important enzymatic processes on earth. In spite of the determination of X-ray structures of increasingly higher resolution, the nitrogenase mechanism is still not understood. In the most recent X-ray structures it has been shown that a carbon resides in the center of the MoFe-cofactor. Its role is not known. Recent spectroscopic studies, mainly EPR, have come closest to obtaining a molecular mechanism for activating nitrogen. Two hydrides have been shown to play a key role in this context. In the present study, the mechanism for nitrogenase has been investigated by hybrid DFT using a cluster model. This approach has been shown to be very successful for predicting mechanisms for other redox-active enzymes, such as the one for photosystem II, but has so far not been used in its most recent form for nitrogenase. The mechanism obtained has large similarities to the one suggested by spectroscopy, with a reductive elimination of two hydrides just before nitrogen binding. However, a very surprising finding is that the central carbon becomes protonated and has to move out of the cavity as a methyl group before the hydrides can be formed. This has not been suggested before.

  2. Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 Å resolution: comparison with the Azotobacter vinelandii MoFe protein

    SciTech Connect

    Zhang, Li-Mei; Morrison, Christine N.; Kaiser, Jens T.; Rees, Douglas C.

    2015-02-01

    Determination of the nitrogenase MoFe protein from C. pasteurianum at 1.08 Å resolution and comparison to its distinct ortholog from A. vinelandii at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

  3. Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

    PubMed Central

    Hernandez, Jose A.; Curatti, Leonardo; Aznar, Constantino P.; Perova, Zinaida; Britt, R. David; Rubio, Luis M.

    2008-01-01

    The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron–molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum–iron–sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex. PMID:18697927

  4. Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium

    PubMed Central

    Fixen, Kathryn R.; Zheng, Yanning; Harris, Derek F.; Shaw, Sudipta; Yang, Zhi-Yong; Dean, Dennis R.; Seefeldt, Lance C.

    2016-01-01

    Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO2 reduction to CH4 in vivo using this remodeled nitrogenase. Conversion of CO2 to CH4 by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO2 in one enzymatic step using light energy. PMID:27551090

  5. Quantitative Geometric Descriptions of the Belt Iron Atoms of the Iron-Molybdenum Cofactor of Nitrogenase and Synthetic Iron(II) Model Complexes

    PubMed Central

    Vela, Javier; Cirera, Jordi; Smith, Jeremy M.; Lachicotte, Rene J.; Flaschenriem, Christine J.; Alvarez, Santiago; Holland, Patrick L.

    2009-01-01

    Six of the seven iron atoms in the iron-molybdenum cofactor of nitrogenase display an unusual geometry, which is distorted from the tetrahedral geometry that is most common in iron-sulfur clusters. This distortion pulls the iron along one C3 axis of the tetrahedron toward a trigonal pyramid. The trigonal pyramidal coordination geometry is rare in four-coordinate transition metal complexes. In order to document this geometry in a systematic fashion in iron(II) chemistry, we have synthesized a range of four-coordinate iron(II) complexes that vary from tetrahedral to trigonal pyramidal. Continuous shape measures are used for a quantitative comparison of the stereochemistry of the Fe atoms in the iron-molybdenum cofactor with those of the presently and previously reported model complexes, as well as with those in polynuclear iron-sulfur compounds. This understanding of the iron coordination geometry is expected to assist in the design of synthetic models for intermediates in the nitrogenase catalytic cycle. PMID:17198413

  6. In-vivo study of the nuclear quadrupole interaction of99Mo (β- 99)Tc in nitrogenase of Klebsiella pneumoniaein nitrogenase of Klebsiella pneumoniae

    NASA Astrophysics Data System (ADS)

    Mottner, P.; Lerf, A.; Ni, X.; Butz, T.; Erfkamp, J.; Müller, A.

    1990-08-01

    We report on the first TDPAC-measurements of the nuclear quadrupole interaction (NQI) of (NQI) of99Mo(β-)99Tc in the nitrogenase of the bacteria Klebsiella pneumoniae. Because nitrogenase is the only Mo-containing enzyme in Klebsiella pneumoniae under the chosen conditions, no further isolation of this enzyme was necessary. The majority of the incorporated99Mo is subjected to a well defined NQI with ω=365(7) Mrad/s, η=1 and a reorientational correlation time of τcoττ≈10nsec and is attributed to the active site of the FeMo cofactor. During sample preparation we noted a pronounced affinity of the bacteria to99mTc.

  7. Large scale isolation and characterization of the molybdenum-iron cluster from nitrogenase.

    PubMed

    Ma, L; Gavini, N; Liu, H I; Hedman, B; Hodgson, K O; Burgess, B K

    1994-07-08

    Here we report the large scale isolation and characterization of a species, designated MoFe cluster, that exhibits an S = 3/2 EPR signal, and the comparison of this entity to isolated FeMo cofactor in N-methylformamide and to the active site of the enzyme nitrogenase. MoFe cluster is isolated from purified nitrogenase by extraction into acidic methyl ethyl ketone and it is stable in that solvent in the absence of thiols. As initially isolated, MoFe cluster solutions exhibit an S = 1/2 EPR signal that arises from an oxidized species that can be reduced by dithionite or thiols to an EPR silent state and then to a state that exhibits an S = 3/2 EPR signal. The S = 3/2 signal is as sharp as the signal exhibited by the protein and much sharper than the signal exhibited by isolated FeMo cofactor. Circular dichroism experiments indicate that unlike the last two species, MoFe cluster does not contain the endogenous ligand R-homocitrate and thus, the sharpness of the S = 3/2 signal is an intrinsic property of the metal center and does not depend upon specific interactions with this organic ligand or with the protein. Metal analyses indicate that the metal core responsible for the S = 3/2 signal contains 6 Fe atoms per molybdenum. X-ray absorption spectroscopy experiments show that although the molybdenum atom in MoFe cluster retains its pseudo-octahedral geometry, its first coordination shell has one less iron atom than that of FeMo cofactor and there has been a significant change in the long range order of the cluster.

  8. The Nitrogenase FeMo-Cofactor and P-Cluster Pair: 2.2 overset{circ}{mathrm A} Resolution Structures

    NASA Astrophysics Data System (ADS)

    Chan, Michael K.; Kim, Jongsun; Rees, D. C.

    1993-05-01

    Structures recently proposed for the FeMo-cofactor and P-cluster pair of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii have been crystallographically verified at 2.2 angstrom resolution. Significantly, no hexacoordinate sulfur atoms are observed in either type of metal center. Consequently, the six bridged iron atoms in the FeMo-cofactor are trigonally coordinated by nonprotein ligands, although there may be some iron-iron bonding interactions that could provide a fourth coordination interaction for these sites. Two of the cluster sulfurs in the P-cluster pair are very close together (~2.1 angstroms), indicating that they form a disulfide bond. These findings indicate that a cavity exists in the interior of the FeMo-cofactor that could be involved in substrate binding and suggest that redox reactions at the P-cluster pair may be linked to transitions of two cluster-bound sulfurs between disulfide and sulfide oxidation states.

  9. Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

    PubMed

    Shah, V K; Rangaraj, P; Chatterjee, R; Allen, R M; Roll, J T; Roberts, G P; Ludden, P W

    1999-05-01

    The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

  10. New structural insights into the iron-molybdenum cofactor from azotobacter vinelandii nitrogenase through sulfur K and molybdenum L x-ray absorption edge studies

    SciTech Connect

    Hedman, B.; Frank, P.; Gheller, S.F.; Roe, A.L.; Newton, W.E.; Hodgson, K.O.

    1988-06-08

    The electronic and structural nature of sulfur and molybdenum in the FeMO cofactor (FeMO-co) isolated from Azotobacter vinelandii MoFe protein has been studied by X-ray absorption edge and near-edge spectroscopy (referred to herein collectively as XANES) at the sulfur K and molybdenum L/sub 3/ and L/sub 2/ absorption edges. In contrast to the relatively poor resolution found for X-ray absorption edges at higher energies (e.g., several electronvolts at the molybdenum K edge at 20 keV), resolution in the 2.5-3.0-keV region is significantly improved (e.g., 0.5 eV at the sulfur K edge at 2.47 keV), resulting in more edge structure with higher sensitivity to changes in electronic and structural environment. In order to record spectra from dilute samples at these low energies, an experimental method that takes advantage of the higher flux synchrotron radiation from an undulator magnet has been developed. XANES spectra have been recorded for FeMo-co in the oxidized (ox) and semireduced (s-r) forms and, for comparison, a number of inorganic complexes containing molybdenum and sulfur. To remove the interference of dithionite, its decomposition products, and other small, unbound molecules from the FeMo-co spectrum, an anaerobic column chromatographic method of purification has been developed. The spectrum of dithionite-free FeMo-co in the oxidized form could thus be recorded.

  11. Iron K-edge X-ray absorption spectroscopy of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae.

    PubMed Central

    Arber, J M; Flood, A C; Garner, C D; Gormal, C A; Hasnain, S S; Smith, B E

    1988-01-01

    Iron K-edge X-ray absorption data for the iron-molybdenum cofactor ('FeMoco') from Klebsiella pneumoniae reported here provide the first evidence for long-range structural order in the cofactor [Fe...Fe(Mo) = 0.368 nm in addition to Fe...S = 0.22 nm and Fe...Fe(Mo) = 0.27 nm] and, in contrast with previously published data [Antonio, Teo, Orme-Johnson, Nelson, Groh, Lindahl, Kauzlarich & Averill (1982) J. Am. Chem. Soc. 104, 4703-4705], indicate that most of the iron centres are not co-ordinated to light (oxygen, nitrogen) atoms. This demonstrates that presently available chemical models for FeMoco are inadequate. PMID:3046607

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

    PubMed

    Guo, Yisong; Echavarri-Erasun, Carlos; Demuez, Marie; Jiménez-Vicente, Emilio; Bominaar, Emile L; Rubio, Luis M

    2016-10-04

    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.

  13. Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase.

    PubMed

    Zhao, Dehua; Curatti, Leonardo; Rubio, Luis M

    2007-12-21

    The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.

  14. Classifying the metal dependence of uncharacterized nitrogenases

    PubMed Central

    McGlynn, Shawn E.; Boyd, Eric S.; Peters, John W.; Orphan, Victoria J.

    2013-01-01

    Nitrogenase enzymes have evolved complex iron–sulfur (Fe–S) containing cofactors that most commonly contain molybdenum (MoFe, Nif) as a heterometal but also exist as vanadium (VFe, Vnf) and heterometal-independent (Fe-only, Anf) forms. All three varieties are capable of the reduction of dinitrogen (N2) to ammonia (NH3) but exhibit differences in catalytic rates and substrate specificity unique to metal type. Recently, N2 reduction activity was observed in archaeal methanotrophs and methanogens that encode for nitrogenase homologs which do not cluster phylogenetically with previously characterized nitrogenases. To gain insight into the metal cofactors of these uncharacterized nitrogenase homologs, predicted three-dimensional structures of the nitrogenase active site metal-cofactor binding subunits NifD, VnfD, and AnfD were generated and compared. Dendrograms based on structural similarity indicate nitrogenase homologs cluster based on heterometal content and that uncharacterized nitrogenase D homologs cluster with NifD, providing evidence that the structure of the enzyme has evolved in response to metal utilization. Characterization of the structural environment of the nitrogenase active site revealed amino acid variations that are unique to each class of nitrogenase as defined by heterometal cofactor content; uncharacterized nitrogenases contain amino acids near the active site most similar to NifD. Together, these results suggest that uncharacterized nitrogenase homologs present in numerous anaerobic methanogens, archaeal methanotrophs, and firmicutes bind FeMo-co in their active site, and add to growing evidence that diversification of metal utilization likely occurred in an anoxic habitat. PMID:23440025

  15. A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents.

    PubMed

    Wink, D A; McLean, P A; Hickman, A B; Orme-Johnson, W H

    1989-11-28

    While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.

  16. Structure and spectroscopy of a bidentate bis-homocitrate dioxo-molybdenum(VI) complex: insights relevant to the structure and properties of the FeMo-cofactor in nitrogenase.

    PubMed

    Zhou, Zhao-Hui; Wang, Hongxin; Yu, Ping; Olmstead, Marilyn M; Cramer, Stephen P

    2013-01-01

    Direct reaction of potassium molybdate (with natural abundance Mo or enriched with (92)Mo or (100)Mo) with excess hydrolyzed homocitric acid-γ-lactone in acidic solution resulted in the isolation of a cis-dioxo bis-homocitrato molybdenum(VI) complex, K(2)[*MoO(2)(R,S-H(2)homocit)(2)]·2H(2)O (1) (*Mo=Mo, 1; (92)Mo, 2; (100)Mo, 3; H(4)homocit=homocitric acid-γ-lactone·H(2)O) and K(2)[MoO(2)((18)O-R,S-H(2)homocit)(2)]·2H(2)O (4). The complex has been characterized by elemental analysis, FT-IR, solid and solution (13)C NMR, and single crystal x-ray diffraction analysis. The molybdenum atom in (1) is quasi-octahedrally coordinated by two cis oxo groups and two bidentate homocitrate ligands. The latter coordinates via its α-alkoxy and α-carboxy groups, while the β- and γ-carboxylic acid groups remain uncomplexed, similar to the coordination mode of homocitrate in the Mo-cofactor of nitrogenase. In the IR spectra, the MoO stretching modes near 900 cm(-1) show 2-3 cm(-1) red- and blue-shifts for the (92)Mo-complex (2) and (100)Mo-complex (3) respectively compared with the natural abundance version (1). At lower frequencies, bands at 553 and 540 cm(-1) are assigned to ν(Mo-O) vibrations involving the alkoxide ligand. At higher frequencies, bands in the 1700-1730 cm(-1) region are assigned to stretching modes of protonated carboxylates. In addition, a band at 1675 cm(-1) was observed that may be analogous to a band seen at 1677 cm(-1) in nitrogenase photolysis studies. The solution behavior of (1) in D(2)O was probed with (1)H and (13)C NMR spectra. An obvious dissociation of homocitrate was found, even though bound to the high valent Mo(VI). This suggests the likely lability of coordinated homocitrate in the FeMo-cofactor with its lower valence Mo(IV).

  17. Nitrogenase assembly

    PubMed Central

    Hu, Yilin; Ribbe, Markus W.

    2013-01-01

    Nitrogenase contains two unique metalloclusters: the P-cluster and the M-cluster. The assembly processes of P- and M-clusters are arguably the most complicated processes in bioinorganic chemistry. There is considerable interest in decoding the biosynthetic mechanisms of the P- and M-clusters, because these clusters are not only biologically important, but also chemically unprecedented. Understanding the assembly mechanisms of these unique metalloclusters is crucial for understanding the structure-function relationship of nitrogenase. Here, we review the recent advances in this research area, with an emphasis on our work that provide important insights into the biosynthetic pathways of these high-nuclearity metal centers. PMID:23232096

  18. Nitrogenase: A Draft Mechanism

    PubMed Central

    Hoffman, Brian M.; Lukoyanov, Dmitriy; Dean, Dennis R.; Seefeldt, Lance C.

    2013-01-01

    Conspectus Biological nitrogen fixation — the reduction of N2 to two NH3 molecules — supports more than half the human population. This reaction is catalyzed by the enzyme nitrogenase, whose predominant form, discussed here, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Nitrogenase has been studied extensively but the catalytic mechanism has remained unknown. At minimum, a mechanism must identify and characterize each intermediate formed during catalysis, and embed these intermediates within a kinetic framework that explains their dynamic interconversion. Nitrogenase kinetics have been described by the Lowe-Thorneley (LT) model, which provides rate constants for transformations among intermediates, denoted En, indexed by the number of electrons (and protons), n, that have been accumulated within the MoFe protein. However, until recently, research on purified nitrogenase had not resulted in characterization of any En state beyond Eo. In this article we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis, and their placement within the LT kinetic scheme. First we discuss the key E4 state, which is primed for N2 binding and reduction and which we refer to as the “Janus intermediate”. This state contains the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C-homocitrate]; FeMo-co) at its resting oxidation level, its four accumulated reducing equivalents being stored as two [Fe-H-Fe] bridging hydrides. The other two trapped intermediates contain reduced forms of N2. One, intermediate I, has S = 1/2 FeMo-co. ENDOR/HYSCORE measurements indicate that I, is the final catalytic state, E8, having NH3 product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (Non-Kramers; S ≥ 2). ESEEM measurements indicate that H binds the [−NH2] fragment and therefore corresponds to E7. These assignments, plus

  19. α-Hydroxy coordination of mononuclear vanadyl citrate, malate and S-citramalate with N−heterocycle ligand, implying a new protonation pathway of iron-vanadium cofactor in nitrogenase

    PubMed Central

    Chen, Can-Yu; Chen, Mao-Long; Chen, Hong-Bin; Wang, Hongxin; Cramer, Stephen P.; Zhou, Zhao-Hui

    2016-01-01

    Unlike the most of α-alkoxy coordination in α-hydroxycarboxylates to vanadium, novel α-hydroxy coordination to vanadium(IV) has been observed for a series of chiral and achiral monomeric α-hydroxycarboxylato vanadyl complexes [VO(H2cit)(bpy)]·2H2O (1), [VO(Hmal)(bpy)]·H2O (2), [VO(H2cit)(phen)]·1.5H2O (3), [VO(Hmal)(phen)]·H2O (4), and [ΔVO(S-Hcitmal)(bpy)]·2H2O (5), [VO(H2cit)(phen)]2·6.5H2O (6), which were isolated from the reactions of vanadyl sulfate with α-hydroxycarboxylates and N-heterocycle ligands in acidic solution. The complexes feature a tridentate citrate, malate or citramalate that chelates to vanadium atom through their α–hydroxy, α–carboxy and β–carboxy groups; while the other β–carboxylic acidic group of citrate is free to participate strong hydrogen bonds with lattice water molecule. The neutral α-hydroxy group also forms strong intermolecular hydrogen bonds with water molecule and the negatively-charged α-carboxy group in the environment. The inclusion of a hydrogen ion in α–alkoxy group results in the formation of a series of neutral complexes with one less positive charge. There are two different configurations of citrate with respect to the trans-position of axial oxo group, where the complex with trans-hydroxy configuration seems more stable with less hindrance. The average bond distances of V–Ohydroxy and V–Oα-carboxy are 2.196 and 2.003 Å respectively, which are comparable to the V–O distance (2.15 Å) of homocitrate in FeV–cofactor of V–nitrogenase. A new structural model is suggested for R-homocitrato iron vanadium cofactor as VFe7S9C(R-Hhomocit) (H4homocit = homocitric acid) with one more proton in homocitrate ligand. PMID:25240212

  20. Cluster assembly in nitrogenase.

    PubMed

    Sickerman, Nathaniel S; Rettberg, Lee A; Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W

    2017-05-09

    The versatile enzyme system nitrogenase accomplishes the challenging reduction of N2and other substrates through the use of two main metalloclusters. For molybdenum nitrogenase, the catalytic component NifDK contains the [Fe8S7]-core P-cluster and a [MoFe7S9C-homocitrate] cofactor called the M-cluster. These chemically unprecedented metalloclusters play a critical role in the reduction of N2, and both originate from [Fe4S4] clusters produced by the actions of NifS and NifU. Maturation of P-cluster begins with a pair of these [Fe4S4] clusters on NifDK called the P*-cluster. An accessory protein NifZ aids in P-cluster fusion, and reductive coupling is facilitated by NifH in a stepwise manner to form P-cluster on each half of NifDK. For M-cluster biosynthesis, two [Fe4S4] clusters on NifB are coupled with a carbon atom in a radical-SAM dependent process, and concomitant addition of a 'ninth' sulfur atom generates the [Fe8S9C]-core L-cluster. On the scaffold protein NifEN, L-cluster is matured to M-cluster by the addition of Mo and homocitrate provided by NifH. Finally, matured M-cluster in NifEN is directly transferred to NifDK, where a conformational change locks the cofactor in place. Mechanistic insights into these fascinating biosynthetic processes are detailed in this chapter. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  1. The Molybdenum Cofactor*

    PubMed Central

    Mendel, Ralf R.

    2013-01-01

    The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. PMID:23539623

  2. The molybdenum cofactor.

    PubMed

    Mendel, Ralf R

    2013-05-10

    The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.

  3. The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

    PubMed

    Dance, Ian

    2015-11-07

    Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, <7 kcal mol(-1). During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The

  4. Nitrogenase and Homologs

    PubMed Central

    2014-01-01

    Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes. PMID:25491285

  5. Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris‡

    PubMed Central

    Oda, Yasuhiro; Samanta, Sudip K.; Rey, Federico E.; Wu, Liyou; Liu, Xiudan; Yan, Tingfen; Zhou, Jizhong; Harwood, Caroline S.

    2005-01-01

    The photosynthetic bacterium Rhodopseudomonas palustris is one of just a few prokaryotes described so far that has vnf and anf genes for alternative vanadium cofactor (V) and iron cofactor (Fe) nitrogenases in addition to nif genes for a molybdenum cofactor (Mo) nitrogenase. Transcriptome data indicated that the 32 genes in the nif gene cluster, but not the anf or vnf genes, were induced in wild-type and Mo nitrogenase-expressing strains grown under nitrogen-fixing conditions in Mo-containing medium. Strains that were unable to express a functional Mo nitrogenase due to mutations in Mo nitrogenase structural genes synthesized functional V and Fe nitrogenases and expressed vnf and anf genes in nitrogen-fixing growth media that contained Mo and V at concentrations far in excess of those that repress alternative nitrogenase gene expression in other bacteria. Thus, not only does R. palustris have multiple enzymatic options for nitrogen fixation, but in contrast to reports on other nitrogen-fixing bacteria, the expression of its alternative nitrogenases is not repressed by transition metals. Between 95 and 295 genes that are not directly associated with nitrogenase synthesis and assembly were induced under nitrogen-fixing conditions, depending on which nitrogenase was being used by R. palustris. Genes for nitrogen acquisition were expressed at particularly high levels during alternative nitrogenase-dependent growth. This suggests that alternative nitrogenase-expressing cells are relatively starved for nitrogen and raises the possibility that fixed nitrogen availability may be the primary signal that controls the synthesis of the V and Fe nitrogenases. PMID:16267302

  6. A New Nitrogenase Mechanism Using a CFe8S9 Model: Does H2 Elimination Activate the Complex to N2 Addition to the Central Carbon Atom?

    PubMed

    McKee, Michael L

    2016-02-11

    A truncated model of the FeMo cofactor is used to explore a new mechanism for the conversion of N2 to NH3 by the nitrogenase enzyme. After four initial protonation/reduction steps, the H4CFe8S9 cluster has two hydrogen atoms attached to sulfur, one hydrogen bridging two iron centers and one hydrogen bonded to carbon. The loss of the CH and FeHFe hydrogens as molecular hydrogen activates the cluster to addition of N2 to the carbon center. This unique step takes place at a nearly planar four-coordinate carbon center and leads to an intermediate with a significantly weakened N-N bond. A hydrogen attached to a sulfur atom is then transferred to the distal nitrogen atom. Additional prontonation/reduction steps are modeled by adding a hydrogen atom to sulfur and locating the transition states for transfer to nitrogen. The first NH3 is lost in a thermal neutral step, while the second step is endothermic. The loss of H2 activates the complex by reducing the barrier for N2 addition by 3.5 kcal/mol. Since this is the most difficult step in the mechanism, reducing the barrier for this step justifies the "extra expense" of H2 production.

  7. A Methanogenic Origin for Molybdenum-Nitrogenase (Invited)

    NASA Astrophysics Data System (ADS)

    Boyd, E.; Miller, S.; Hamilton, T.; Lavin, M.; Peters, J.

    2009-12-01

    The taxonomic distribution and phylogenetic relationships of proteins required for molybdenum (Mo)-nitrogenase that arose by gene fusion and duplication reveals that Mo-nitrogenase was not associated with LUCA, but rather emerged in the strictly anaerobic methanogenic archaea and was acquired in bacteria via lateral gene transfer in an anoxic environment. Therefore, it was hypothesized that Mo-nitrogenase emerged early during the evolution of life, perhaps prior to the emergence of oxygenic photosynthesis. To test this hypothesis, we examined the evolutionary relationships of paralogous proteins required for the biosynthesis of the nitrogenase active site cofactor and bacteriochlorophyll (Bch), which indicated that Mo-nitrogenase predates the emergence of oxygenic photosynthesis. Importantly, the age of nodes delineating the major diversification of Mo-dependent nitrogenase is similar to the maximum age for the emergence of oxygenic photosynthesis, suggesting that the diversification of Mo-nitrogenase may have been promoted by the emergence of oxygenic photosynthesis, most likely through the widespread oxidation of Mo-sulfides and subsequent increases in Mo bioavailability. These findings imply that Mo-dependent biological nitrogen fixation emerged prior to the transition from the Archean to the Proterozoic and the widespread oxidation of the atmosphere and ocean. Further, the results imply that the emergence and evolution of biological nitrogen fixation is closely tied to the evolution of the redox of the global biosphere.

  8. Molybdenum Enzymes, Cofactors, and Model Systems.

    ERIC Educational Resources Information Center

    Burgmayer, S. J. N; Stiefel, E. I.

    1985-01-01

    Discusses: (l) molybdoenzymes (examining their distribution and metabolic role, composition and redox strategy, cofactors, substrate reactions, and mechanistic possibilities); (2) structural information on molybdenum (Mo) centers; (3) modeling studies (Mo-co models, nitrogenase models, and the MO-S duo); and (4) the copper-molybdenum antagonism.…

  9. Molybdenum Enzymes, Cofactors, and Model Systems.

    ERIC Educational Resources Information Center

    Burgmayer, S. J. N; Stiefel, E. I.

    1985-01-01

    Discusses: (l) molybdoenzymes (examining their distribution and metabolic role, composition and redox strategy, cofactors, substrate reactions, and mechanistic possibilities); (2) structural information on molybdenum (Mo) centers; (3) modeling studies (Mo-co models, nitrogenase models, and the MO-S duo); and (4) the copper-molybdenum antagonism.…

  10. Fe and Mo EXAFS of Azotobacter vinelandii nitrogenase inpartially oxidized and singly reduced forms

    SciTech Connect

    Christiansen, J.; Tittsworth, R.C.; Hales, B.J.; Cramer, S.P. |

    1995-10-11

    Fe and Mo K-edge EXAFS spectra of the nitrogenase Mo-Fe protein in the indigo disulfonate (IDS) oxidized form and under slow turnover conditions have been recorded. The EXAFS of the one-electron reduced form E{sub 1} was obtained as a difference spectrum between the slow turnover and resting (E{sub 0}) spectra. Average Fe-S, Fe-Fe, and Fe-Mo distances of 2.33, 2.60, and 2.66A, respectively, along with a second Fe-Fe distance at 3.72 A were found for E{sub 1}. The IDS-oxidized MoFe protein contain spartially oxidized `P-clusters`. For this sample, average Fe-S,Fe-Fe, and Fe-Mo interactions at 2.31, 2.65, and 2.71 A, respectively, were found along with the long Fe-Fe interaction at 3.74 A. Combination of the current results with previous data on resting and thionin-oxidized nitrogenase shows a general trend a significant number of the metal-metal distances tend to contract as the enzyme becomes more reduced. 52 refs., 6 figs., 2 tabs.

  11. Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

    SciTech Connect

    Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C.

    2014-03-22

    Mo-dependent nitrogenase catalyzes the biological reduction of N2 to 2NH3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel not previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

  12. Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments

    PubMed Central

    McRose, Darcy L.; Zhang, Xinning; Kraepiel, Anne M. L.; Morel, François M. M.

    2017-01-01

    The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient ‘canonical’ Mo-nitrogenase, whereas Fe-only and V-(‘alternative’) nitrogenases are often considered ‘backup’ enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation. PMID:28293220

  13. X-ray absorption of Azotobacter vinelandii vanadium nitrogenase

    SciTech Connect

    George, G.N.; Coyle, C.L.; Hales, B.J.; Cramer, S.P.

    1988-06-08

    Evidence for the existence of a vanadium-containing nitrogenase has existed for more than half a century, but progress in understanding this enzyme has only come recently. In 1980, Bishop and co-workers proposed that an alternative nitrogen-fixing enzyme exists in Azotobacter vinelandii and subsequently proposed that vanadium was involved. In 1986, Robson et al. demonstrated clearly that the alternate nitrogenase from Azotobacter chroococcum, Acl*, contained vanadium instead of molybdenum. Hales et al. have shown the vanadium is also found in the Azotobacter vinelandii alternative component I, Avl'. The molybdenum and vanadium nitrogenase proteins are similar in many respects. Like the molybdenum enzyme, both Acl* and Avl' exhibit an EPR spectrum characteristic of a species with an S = 3/2 ground state; Avl' also contains the so-called P-clusters. Additionally Acl* has recently been shown to possess an N-methylformamide soluble cofactor, FeVco, analogous to the well-known iron-molybdenum cofactor FeMoco. Arber et al. have reported X-ray absorption spectra for the Acl* enzyme and interpreted the EXAFS as evidence for a V-Fe-S cluster. The local vanadium structure is proposed to resemble a recently synthesized cubane-like VFe/sub 3/S/sub 4/ cluster, and analogies are drawn with the EXAFS-derived structure reported for the molybdenum nitrogenases. The authors report herein an X-ray absorption spectroscopic study of A. vinelandii vanadium nitrogenase, Avl', which supports and extends the work of Arber et al.

  14. Posttranslational modification of a vanadium nitrogenase.

    PubMed

    Heiniger, Erin K; Harwood, Caroline S

    2015-08-01

    In microbes that fix nitrogen, nitrogenase catalyzes the conversion of N2 to ammonia in an ATP-demanding reaction. To help conserve energy some bacteria inhibit nitrogenase activity upon exposure to ammonium. The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 can synthesize three functional nitrogenase isoenzymes: a molybdenum nitrogenase, a vanadium nitrogenase, and an iron nitrogenase. Previous studies showed that in some alphaproteobacteria, including R. palustris, molybdenum nitrogenase activity is inhibited by ADP-ribosylation when cells are exposed to ammonium. Some iron nitrogenases are also posttranslationally modified. However, the posttranslational modification of vanadium nitrogenase has not been reported. Here, we investigated the regulation of the alternative nitrogenases of R. palustris and determined that both its vanadium nitrogenase and its iron nitrogenase activities were inhibited and posttranslationally modified when cells are exposed to ammonium. Vanadium nitrogenase is not found in all strains of R. palustris, suggesting that it may have been acquired by horizontal gene transfer. Also, phylogenetic analyses of the three nitrogenases suggest that VnfH, the target of ADP-ribosylation, may be the product of a gene duplication of nifH, the molybdenum nitrogenase homolog.

  15. Nitrogenase Assembly: Strategies and Procedures.

    PubMed

    Sickerman, Nathaniel S; Hu, Yilin; Ribbe, Markus W

    2017-01-01

    Nitrogenase is a metalloenzyme system that plays a critical role in biological nitrogen fixation, and the study of how its metallocenters are assembled into functional entities to facilitate the catalytic reduction of dinitrogen to ammonia is an active area of interest. The diazotroph Azotobacter vinelandii is especially amenable to culturing and genetic manipulation, and this organism has provided the basis for many insights into the assembly of nitrogenase proteins and their respective metallocofactors. This chapter will cover the basic procedures necessary for growing A. vinelandii cultures and subsequent recombinant transformation and protein expression techniques. Furthermore, protocols for nitrogenase protein purification and substrate reduction activity assays are described. These methods provide a solid framework for the assessment of nitrogenase assembly and catalysis. © 2017 Elsevier Inc. All rights reserved.

  16. Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster.

    PubMed Central

    Robinson, A C; Burgess, B K; Dean, D R

    1986-01-01

    The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed. Images PMID:3457004

  17. Iron EXAFS of Azotobacter vinelandii nitrogenase Mo-Fe and V-Fe proteins

    SciTech Connect

    Chen, J. ); Christiansen, J.; George, S.J. ); Tittsworth, R.C.; Hales, B.J. ); Concouvanis, D. ); Cramer, S.P. Univ. of California, Davis )

    1993-06-30

    The structure of the iron sites of nitrogenase in dithionite-reduced and thionine-oxidized forms of the Mo-Fe and V-Fe proteins has been investigated using Fe K-edge X-ray absorption spectroscopy. For the dithionite-reduced Azotobacter vinelandii Mo-Fe protein, the dominant EXAFS Fourier transform peaks are assigned to Fe-S and Fe-Fe interactions at approximately 2.32 and 2.64 [angstrom], as expected for Fe-S clusters. An additional Fe-Mo component at 2.73 [angstrom] is required to completely fit the EXAFS in the 1-3-[angstrom] region. In the 3-5-[angstrom] region, a 3.8-[angstrom] Fe-Fe component is identified, with an amplitude corresponding to almost one long Fe-Fe interaction, averaged over all of the iron in the sample. Features that can be explained as Fe-S and Fe-Fe interactions at 4.3 and 4.7 [angstrom] are also observed. A similar pattern of Fe interactions is observed for the reduced A. vinelandii V-Fe protein, except that the short Fe-Mo interaction is no longer required. In both Mo-Fe and V-Fe proteins, the first coordination sphere Fe-S distances contract slightly upon thionine oxidation. The long-range Fe-S and Fe-Fe interactions are very close (within 0.1 [angstrom]) to corresponding distances in Fe[sub 6]S[sub 6] prismane clusters. If the amplitudes are adjusted by assuming that only 14 of 30 nitrogenase irons participate in the M center, then they are consistent with recently proposed crystallographic models. 43 refs., 7 figs., 2 tabs.

  18. Interspecies homology of nitrogenase genes.

    PubMed Central

    Ruvkun, G B; Ausubel, F M

    1980-01-01

    Cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species. K. pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K. pneumoniae 3 x 10(9) years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time. Images PMID:6987649

  19. Structural Models for the Metal Centers in the Nitrogenase Molybdenum-Iron Protein

    NASA Astrophysics Data System (ADS)

    Kim, Jongsun; Rees, D. C.

    1992-09-01

    Structural models for the nitrogenase FeMo-cofactor and P-clusters are proposed based on crystallographic analysis of the nitrogenase molybdenum-iron (MoFe)-protein from Azotobacter vinelandii at 2.7 angstrom resolution. Each center consists of two bridged clusters; the FeMo-cofactor has 4Fe:3S and 1Mo:3Fe:3S clusters bridged by three non-protein ligands, and the P-clusters contain two 4Fe:4S clusters bridged by two cysteine thiol ligands. Six of the seven Fe sites in the FeMo-cofactor appear to have trigonal coordination geometry, including one ligand provided by a bridging group. The remaining Fe site has tetrahedral geometry and is liganded to the side chain of Cys^α275. The Mo site exhibits approximate octahedral coordination geometry and is liganded by three sulfurs in the cofactor, two oxygens from homocitrate, and the imidazole side chain of His^α442. The P-clusters are liganded by six cysteine thiol groups, two which bridge the two clusters, α88 and β95, and four which singly coordinate the remaining Fe sites, α62, α154, β70, and β153. The side chain of Ser^β188 may also coordinate one iron. The polypeptide folds of the homologous α and β subunits surrounding the P-clusters are approximately related by a twofold rotation that may be utilized in the binding interactions between the MoFe-protein and the nitrogenase Fe-protein. Neither the FeMo-cofactor nor the P-clusters are exposed to the surface, suggesting that substrate entry, electron transfer, and product release must involve a carefully regulated sequence of interactions between the MoFe-protein and Fe-protein of nitrogenase.

  20. The role of X-ray spectroscopy in understanding the geometric and electronic structure of nitrogenase.

    PubMed

    Kowalska, Joanna; DeBeer, Serena

    2015-06-01

    X-ray absorption (XAS) and X-ray emission spectroscopy (XES) provide element specific probes of the geometric and electronic structures of metalloprotein active sites. As such, these methods have played an integral role in nitrogenase research beginning with the first EXAFS studies on nitrogenase in the late 1970s. Herein, we briefly explain the information that can be extracted from XAS and XES. We then highlight the recent applications of these methods in nitrogenase research. The influence of X-ray spectroscopy on our current understanding of the atomic structure and electronic structure of iron molybdenum cofactor (FeMoco) is emphasized. Contributions of X-ray spectroscopy to understanding substrate interactions and cluster biosynthesis are also discussed. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  1. A molecular pathway for the egress of ammonia produced by nitrogenase

    NASA Astrophysics Data System (ADS)

    Dance, Ian

    2013-11-01

    Nitrogenase converts N2 to NH3, at one face of an Fe-Mo-S cluster (FeMo-co) buried in the protein. Through exploration of cavities in the structures of nitrogenase proteins, a pathway for the egress of ammonia from its generation site to the external medium is proposed. This pathway is conserved in the three species Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteurianum. A molecular mechanism for the translocation of NH3 by skipping through a sequence of hydrogen bonds involving eleven water molecules and surrounding aminoacids has been developed. The putative mechanism requires movement aside of some water molecules by up to ~ 1Å. Consistent with this, the surrounding protein is comprised of different chains and has little secondary structure: protein fluctuations are part of the mechanism. This NH3 pathway is well separated from the water chain and embedded proton wire that have been proposed for serial supply of protons to FeMo-co. Verification procedures are suggested.

  2. Refining the pathway of carbide insertion into the nitrogenase M-cluster

    PubMed Central

    Wiig, Jared A.; Hu, Yilin; Ribbe, Markus W.

    2015-01-01

    Carbide insertion plays a pivotal role in the biosynthesis of M-cluster, the cofactor of nitrogenase. Previously, we proposed a carbide insertion pathway involving methyltransfer from SAM to a FeS precursor and hydrogen abstraction from this methyl group that initiates the radical-based precursor maturation. Here we demonstrate that the methyl group is transferred to a precursor-associated sulfur before hydrogen abstraction, thereby refining the initial steps of the carbide insertion pathway. PMID:26259825

  3. Precipitation phase transformation in nanocrystalline Fe-Mo alloys.

    PubMed

    Sarkar, Subhajit; Bansal, Chandrahaas

    2004-01-01

    Precipitation phase transformation was studied in nanocrystalline Fe-rich Fe-Mo alloys with the use of X-ray diffraction and Mössbauer spectroscopy. Alloys up to 5 at% Mo in Fe were synthesized by mechanical alloying and formed in alpha phase bcc solid solutions with average grain sizes in the range of 10-13 nm. The precipitation transformation (alpha-->alpha + lambda) was found to proceed via a Mo clustering that was correlated with the size of the nanograins. This was understood in terms of the Gibbs Thomson effect with a concept of negative surface energy contribution to the Gibbs free energy of mixing in a nanocrystalline alloy with positive internal energy of mixing. This contribution increased the stability of the solid solution for nanosized grains, and the Mo precipitation started once the grains grew beyond a critical size. We argue that the Mo precipitation takes place in the grain boundary regions, and the Mo-rich lambda phase also precipitates directly in the grain boundary regions, in contrast to the microcrystalline alloys, where the Mo clusters formed within the grains and were first dissolved in the Fe matrix before the lambda phase was formed.

  4. The Role of Oxygen in the Evolution of Molybdenum Nitrogenase

    NASA Astrophysics Data System (ADS)

    Peters, J.; Boyd, E. S.; Hamilton, T. L.

    2012-12-01

    Since early in Earth's history, the supply of nitrogen (N) into the biosphere has been controlled by the activity of nitrogenase, an oxygen sensitive enzyme that catalyzes the reduction of dinitrogen gas (N2) to bioavailable ammonia. The most common form of nitrogenase harbors a complex molybdenum (Mo) cofactor at its active site [Mo-nitrogenase (Nif)], although other phylogenetically related (alternative) forms of nitrogenase that differ in their active-site metal composition also likely contribute NH3 in environments that are limiting in Mo. The solubility of Mo is significantly influenced by redox and this fact has been used to argue that that the iron (Fe)-dependent nitrogenase (Anf) was predominant prior to ~ 2.5 Ga because oceans were depleted in Mo and rich in Fe. This hypothesis, however, is inconsistent with recent phylogenetic data which strongly suggest that Anf is derived from Nif. Here, we examine the evolutionary history of the Nif enzyme complex in reference to the physiological, biochemical, and morphological strategies for reducing damage by molecular oxygen. A total of 189 nif operons were characterized and quantitatively mapped on a NifHDK concatenated phylogenetic tree. An overlay of the primary mode of metabolism, as defined as either anaerobic (AN) or aerobic/facultative aerobic (AFA), on the NifHDK tree indicates that Nif originated in an anoxic environment and was first acquired in an AFA lineage within the actinobacteria. The complexity of nif operons increased during the evolutionary history of Nif, with a pronounced increase observed during the transition from AN to AFA modes of metabolism. This increase in operon complexity is accompanied by a number of gene loss (nifI1 and nifI2) and gene acquisition (nifW, nifT, nifZ, nifQ) events, with variation in the overall composition of nif operons attributable to adaptations that mediated the toxicity of O2. Collectively, these results underscore the role of O2 in shaping the evolutionary

  5. Docking and Migration of Carbon Monoxide in Nitrogenase: The Case for Gated Pockets from IR Spectroscopy and Molecular Dynamics

    PubMed Central

    Gee, Leland B.; Leontyev, Igor; Stuchebrukhov, Alexei; Scott, Aubrey D.; Pelmenschikov, Vladimir; Cramer, Stephen P.

    2015-01-01

    Evidence for a CO docking site near the FeMo-cofactor in nitrogenase has been obtained by FT-IR monitored low temperature photolysis. We investigated the possible migration paths for CO from this docking site using molecular dynamics calculations. The simulations support the notion of a gas channel with multiple internal pockets from the active site to the protein exterior. Travel between pockets is gated by motion of protein residues. Implications for the mechanism of nitrogenase reactions with CO and N2 are discussed. PMID:25919807

  6. Biosynthesis and Insertion of the Molybdenum Cofactor.

    PubMed

    Magalon, Axel; Mendel, Ralf R

    2015-01-01

    The transition element molybdenum (Mo) is of primordial importance for biological systems, because it is required by enzymes catalyzing key reactions in the global carbon, sulfur, and nitrogen metabolism. To gain biological activity, Mo has to be complexed by a special cofactor. With the exception of bacterial nitrogenase, all Mo-dependent enzymes contain a unique pyranopterin-based cofactor coordinating a Mo atom at their catalytic site. Various types of reactions are catalyzed by Mo-enzymes in prokaryotes including oxygen atom transfer, sulfur or proton transfer, hydroxylation, or even nonredox reactions. Mo-enzymes are widespread in prokaryotes and many of them were likely present in the Last Universal Common Ancestor. To date, more than 50--mostly bacterial--Mo-enzymes are described in nature. In a few eubacteria and in many archaea, Mo is replaced by tungsten bound to the same unique pyranopterin. How Mo-cofactor is synthesized in bacteria is reviewed as well as the way until its insertion into apo-Mo-enzymes.

  7. Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component

    PubMed Central

    Fay, Aaron W.; Blank, Michael A.; Rebelein, Johannes G.; Lee, Chi Chung; Ribbe, Markus W.; Hedman, Britt; Hodgson, Keith O.; Hu, Yilin

    2016-01-01

    NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster–bound NifEN (designated NifENM) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifENM upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifENM with an M-cluster in one αβ-half and an empty cluster-binding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifENM displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster–bound αβ-dimer of NifENM as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase. PMID:27506795

  8. Vanadium nitrogenase: a two-hit wonder?

    PubMed

    Hu, Yilin; Lee, Chi Chung; Ribbe, Markus W

    2012-01-28

    Nitrogenase catalyzes the biological conversion of atmospheric dinitrogen to bioavailable ammonia. The molybdenum (Mo)- and vanadium (V)-dependent nitrogenases are two homologous members of this metalloenzyme family. However, despite their similarities in structure and function, the characterization of V-nitrogenase has taken a much longer and more winding path than that of its Mo-counterpart. From the initial discovery of this nitrogen-fixing system, to the recent finding of its CO-reducing capacity, V-nitrogenase has proven to be a two-hit wonder in the over-a-century-long research of nitrogen fixation. This perspective provides a brief account of the catalytic function and structural basis of V-nitrogenase, as well as a short discussion of the theoretical and practical potentials of this unique metalloenzyme.

  9. Vanadium Nitrogenase: A Two-Hit Wonder?

    PubMed Central

    Hu, Yilin; Lee, Chi Chung; Ribbe, Markus W.

    2013-01-01

    Nitrogenase catalyzes the biological conversion of atmospheric dinitrogen to bioavailable ammonia. The molybdenum (Mo)- and vanadium (V)-dependent nitrogenases are two homologous members of this metalloenzyme family. However, despite their similarities in structure and function, the characterization of V-nitrogenase has taken a much longer and more winding path than that of its Mo-counterpart. From the initial discovery of this nitrogen-fixing system, to the recent finding of its CO-reducing capacity, V-nitrogenase has proven to be a two-hit wonder in the over-a-century-long research of nitrogen fixation. This perspective provides a brief account of the catalytic function and structural basis of V-nitrogenase, as well as a short discussion of the theoretical and practical potentials of this unique metalloenzyme. PMID:22101422

  10. EXAFS of Klebsiella pneumoniae nitrogenase MoFe protein from wild-type and nif V mutant strains

    SciTech Connect

    Eidsness, M.K.; Flank, A.M.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer. S.P.

    1986-05-14

    The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiella pneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

  11. Metal and cofactor insertion.

    PubMed

    Mendel, Ralf R; Smith, Alison G; Marquet, Andree; Warren, Martin J

    2007-10-01

    Cells require metal ions as cofactors for the assembly of metalloproteins. Principally one has to distinguish between metal ions that are directly incorporated into their cognate sites on proteins and those metal ions that have to become part of prosthetic groups, cofactors or complexes prior to insertion of theses moieties into target proteins. Molybdenum is only active as part of the molybdenum cofactor, iron can be part of diverse Fe-S clusters or of the heme group, while copper ions are directly delivered to their targets. We will focus in greater detail on molybdenum metabolism because molybdenum metabolism is a good example for demonstrating the role and the network of metals in metabolism: each of the three steps in the pathway of molybdenum cofactor formation depends on a different metal (iron, copper, molybdenum) and also the enzymes finally harbouring the molybdenum cofactor need additional metal-containing groups to function (iron sulfur-clusters, heme-iron).

  12. Possible evolutionary relationships of the nitrogenase proteins.

    PubMed

    Kleiner, D; Littke, W; Bender, H; Wallenfels, K

    1976-03-29

    Comparisons of the amino acid compositions of the nitrogenase proteins from different organisms and their correlation with cross-reactivities and taxonomical data suggest an evolution within bacterial genomes rather than within plasmids. Comparisons of the amino acid compositions of nitrogenases and other ATP-ases show similarities which might be due to the evolution of these ATP-ases from a common ancestral protein.

  13. Surface, optical characteristics and photocatalytic ability of Scheelite-type monoclinic Bi3FeMo2O12 nanoparticles

    NASA Astrophysics Data System (ADS)

    Nie, Xinming; Wulayin, Wumitijiang; Song, Tingting; Wu, Minxiao; Qiao, Xuebin

    2016-11-01

    Bi3FeMo2O12 nanoparticles with the Scheelite-type monoclinic structure were prepared by the Pechini synthesis. The Bi3FeMo2O12 nanoparticle has a size of about 50 nm. The phase formation and structural characteristic were studied by X-ray diffraction (XRD) patterns and Rietveld refinements. The Scheelite framework is characterized by a superstructure constructed by the ordered arrangement of Fe/Mo tetrahedral on the B sites. The surface characteristics of Bi3FeMo2O12 nanoparticles were studied by the measurements such as the scanning electron microscope (SEM), the transmission electron microscopy (TEM), and the N2-adsorption-desorption isotherm. Bi3FeMo2O12 nanoparticles present an efficient optical absorption in a wide wavelength region from UV to 540 nm. The band gap energy was decided to be 2.3 eV and characterized by a direct allowed electronic optical transition. The photocatalytic activity of Bi3FeMo2O12 nanoparticles was confirmed by the photodegradation of the rhodamine B (RhB) dye solution. The experiments indicate that the Scheelite-type molybdate could be a potential candidate of a visible-light-driven photocatalyst.

  14. Using synthetic biology to increase nitrogenase activity.

    PubMed

    Li, Xin-Xin; Liu, Qi; Liu, Xiao-Meng; Shi, Hao-Wen; Chen, San-Feng

    2016-02-20

    Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe-S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ can not increase activity. The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.

  15. Nitrite, a new substrate for nitrogenase

    SciTech Connect

    Vaughn, S.A.; Burgess, B.K. )

    1989-01-24

    The authors have examined the reactivity of the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2) toward nitrate and nitrite. Nitrate has no effect on H{sub 2} evolution or C{sub 2}H{sub 2} reduction by nitrogenase and thus is neither a substrate nor an inhibitor. Nitrite dramatically inhibits H{sub 2} evolution. This inhibition has two components, one irreversible and one reversible upon addition of CO. The irreversible inhibition is due to nitrite inactivation of the Fe protein. The rate of this inactivation is greatly enhanced by addition of MgATP, suggesting the (4Fe-4S) cluster is the site of nitrite attack. The reversible inhibition does not represent an inhibition of electron flow but rather a diversion of electrons away from H{sub 2} evolution and into the six-electron reduction of nitrite to ammonia. Thus, nitrogenase functions as a nitrite reductase.

  16. The biosynthesis of the molybdenum cofactors.

    PubMed

    Mendel, Ralf R; Leimkühler, Silke

    2015-03-01

    The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.

  17. Fe Protein-Independent Substrate Reduction by Nitrogenase MoFe Protein Variants

    SciTech Connect

    Danyal, Karamatullah; Rasmussen, Andrew J.; Keable, Stephen M.; Inglet, Boyd S.; Shaw, Sudipta; Zadvornyy, Oleg; Duval, Simon S.; Dean, Dennis R.; Raugei, Simone; Peters, John W.; Seefeldt, Lance C.

    2015-04-21

    The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo-cofactor. Here, it is reported that independent substitution of three amino acids (ß-98Tyr→His, α-64Tyr→His, and ß-99Phe→His) located between the P cluster and FeMo-cofactor within the MoFe protein endows it with the ability to reduce protons to H2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low potential reductant polyaminocarboxylate ligated Eu(II) (Em -1.1 to -0.84 V vs NHE). The crystal structure of the ß-98Tyr→His variant MoFe protein was determined, revealing only small changes near the amino acid substitution that affect the solvent structure and immediate vicinity between the P cluster and the FeMo-cofactor, with no global conformational changes observed. Computational normal mode analysis on the nitrogenase complex reveal coupling in the motions of the Fe protein and the region of the MoFe protein with these three amino acids, which suggests a possible mechanism for how Fe protein might communicate deep within the MoFe protein subtle changes that profoundly affect intramolecular electron transfer and substrate reduction. This work was supported by a grant from the National Science Foundation (MCB-1330807) to JWP and LCS. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (DE-SC0010687 and DE-SC0010834 to LCS and DRD) and the Division of Chemical Sciences, Geosciences, and Bio-Sciences (SR). The coordinates for the ß-98His MoFe protein were deposited with the Protein Data Bank (PDB 4XPI).

  18. Crystallographic studies of nitrogenase and hydrogenase

    NASA Astrophysics Data System (ADS)

    Bolin, J. T.

    The long term goal of this project is to obtain detailed knowledge of the structure and function of nitrogenase and hydrogenase through the analysis of physical, chemical, and biological data with reference to three-dimensional, atomic resolution crystal structures of components of the enzyme and/or complexes of the components. The current objectives to determine the crystal structure of wild-type Av1, the nitrogenase MoFe protein from Azotobacter vinelandii; to refine this structure at high resolution; and to initiate studies of mutant MoFe proteins that express altered chemical and physical properties. Further we seek to determine the crystal structure of the bi-directional all-Fe hydrogenase from C. pasteurianum, Cp-hydrI, and to initiate studies of the uptake hydrogenase from the same organism, Cp-hydrii.

  19. Multiple Amino Acid Sequence Alignment Nitrogenase Component 1: Insights into Phylogenetics and Structure-Function Relationships

    PubMed Central

    Howard, James B.; Kechris, Katerina J.; Rees, Douglas C.; Glazer, Alexander N.

    2013-01-01

    Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as “core” for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases

  20. Multiple amino acid sequence alignment nitrogenase component 1: insights into phylogenetics and structure-function relationships.

    PubMed

    Howard, James B; Kechris, Katerina J; Rees, Douglas C; Glazer, Alexander N

    2013-01-01

    Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as "core" for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases for

  1. The controlled relay of multiple protons required at the active site of nitrogenase.

    PubMed

    Dance, Ian

    2012-07-07

    The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

  2. Autonomous Filling of Grain-Boundary Cavities during Creep Loading in Fe-Mo Alloys

    NASA Astrophysics Data System (ADS)

    Zhang, S.; Fang, H.; Gramsma, M. E.; Kwakernaak, C.; Sloof, W. G.; Tichelaar, F. D.; Kuzmina, M.; Herbig, M.; Raabe, D.; Brück, E.; van der Zwaag, S.; van Dijk, N. H.

    2016-10-01

    We have investigated the autonomous repair of creep damage by site-selective precipitation in a binary Fe-Mo alloy (6.2 wt pct Mo) during constant-stress creep tests at temperatures of 813 K, 823 K, and 838 K (540 °C, 550 °C, and 565 °C). Scanning electron microscopy studies on the morphology of the creep-failed samples reveal irregularly formed deposits that show a close spatial correlation with the creep cavities, indicating the filling of creep cavities at grain boundaries by precipitation of the Fe2Mo Laves phase. Complementary transmission electron microscopy and atom probe tomography have been used to characterize the precipitation mechanism and the segregation at grain boundaries in detail.

  3. Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia.

    PubMed

    Zhang, Xinning; Sigman, Daniel M; Morel, François M M; Kraepiel, Anne M L

    2014-04-01

    Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth's ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ(15)N) of newly fixed N is currently believed to be ∼-1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only "alternative" nitrogenases produce fixed N with significantly lower δ(15)N (-6 to -7‰). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ(15)N (<-2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen.

  4. Biology of the molybdenum cofactor.

    PubMed

    Mendel, Ralf R

    2007-01-01

    The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.

  5. Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.

    PubMed

    Yan, Lifen; Dapper, Christie H; George, Simon J; Wang, Hongxin; Mitra, Devrani; Dong, Weibing; Newton, William E; Cramer, Stephen P

    2011-05-01

    Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm(-1) and 1679 cm(-1) together with positive bands at 1711 cm(-1), 2135 and 2123 cm(-1). The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm(-1) is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol(-1). Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm(-1) with a positive 'Lo-2' band at 1780 cm(-1), while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm(-1) and a positive feature 'Lo-3' band at 1921 cm(-1). These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

  6. FEMO, A FLOW AND ENRICHMENT MONITOR FOR VERIFYING COMPLIANCE WITH INTERNATIONAL SAFEGUARDS REQUIREMENTS AT A GAS CENTRIFUGE ENRICHMENT FACILITY

    SciTech Connect

    Gunning, John E; Laughter, Mark D; March-Leuba, Jose A

    2008-01-01

    A number of countries have received construction licenses or are contemplating the construction of large-capacity gas centrifuge enrichment plants (GCEPs). The capability to independently verify nuclear material flows is a key component of international safeguards approaches, and the IAEA does not currently have an approved method to continuously monitor the mass flow of 235U in uranium hexafluoride (UF6) gas streams. Oak Ridge National Laboratory is investigating the development of a flow and enrichment monitor, or FEMO, based on an existing blend-down monitoring system (BDMS). The BDMS was designed to continuously monitor both 235U mass flow and enrichment of UF6 streams at the low pressures similar to those which exists at GCEPs. BDMSs have been installed at three sites-the first unit has operated successfully in an unattended environment for approximately 10 years. To be acceptable to GCEP operators, it is essential that the instrument be installed and maintained without interrupting operations. A means to continuously verify flow as is proposed by FEMO will likely be needed to monitor safeguards at large-capacity plants. This will enable the safeguards effectiveness that currently exists at smaller plants to be maintained at the larger facilities and also has the potential to reduce labor costs associated with inspections at current and future plants. This paper describes the FEMO design requirements, operating capabilities, and development work required before field demonstration.

  7. Nitrogenase activity of immobilized Azotobacter vinelandii.

    PubMed

    Seyhan, E; Kirwan, D J

    1979-02-01

    As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotobacter vinelandii was undertaken. Immobilization was acaccomplished by adsorption onto an anionic exchange cellulose (Cellex E) with loadings as high as 10'' cells/g resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenase activities as high as 4200 nmol/min g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.

  8. Biosynthesis of Nitrogenase FeMoco

    PubMed Central

    Hu, Yilin; Ribbe, Markus W.

    2010-01-01

    Biosynthesis of nitrogenase FeMoco is a highly complex process that requires, minimally, the participation of nifS, nifU, nifB, nifE, nifN, nifV, nifH, nifD and nifK gene products. Previous genetic analyses have identified the essential factors for the assembly of FeMoco; however, the exact functions of these factors and the precise sequence of events during the assembly process had remained unclear until recently, when a number of the biosynthetic intermediates of FeMoco were identified and characterized by combined biochemical, spectroscopic and structural analyses. This review gives a brief account of the recent progress toward understanding the assembly process of FeMoco, which has identified some important missing pieces of this biosynthetic puzzle. PMID:21503270

  9. Estimation of Nitrogenase Using a Colorimetric Determination for Ethylene 1

    PubMed Central

    Larue, T. A.; Kurz, W. G. W.

    1973-01-01

    Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene. PMID:16658468

  10. Experimental Investigation and Computer Simulation of Diffusion in Fe-Mo and Fe-Mn-Mo Alloys with Different Optimization Methods

    NASA Astrophysics Data System (ADS)

    Zheng, Weisen; Ågren, John; Lu, Xiao-Gang; He, Yanlin; Li, Lin

    2017-01-01

    In order to simulate the diffusional phase transformations involving the fcc and bcc phases for microalloyed steels, the diffusion mobilities for fcc and bcc Fe-Mo and Fe-Mn-Mo alloys were experimentally investigated and critically assessed. The diffusion-couple technique was employed to extract the interdiffusion coefficients in Fe-Mo and Fe-Mn-Mo alloys with the Sauer-Freise and Whittle-Green methods. Based on the present experimental interdiffsivities, the mobility parameters for the fcc and bcc phases in the Fe-Mo and Fe-Mn-Mo systems were optimized using the traditional method. Simultaneously, a direct method was developed and utilized to directly fit mobilities to the diffusion profiles rather than the diffusivities in the present work. The satisfactory description of the diffusion behavior in the Fe-Mo and Fe-Mn-Mo systems has confirmed the reliability of the direct method. Particularly, the two sets of diffusion mobilities obtained with both methods could simulate the diffusion phenomenon between the fcc and bcc phases in the Fe-Mo and Fe-Mn-Mo systems successfully.

  11. Why should we consider alternative nitrogenases in boreal ecosystems?

    NASA Astrophysics Data System (ADS)

    Bellenger, Jean-Philippe

    2014-05-01

    Biological nitrogen fixation (BNF) is the main source of new nitrogen (N) for the biosphere, accounting for up to 97% of N input in unmanaged terrestrial ecosystems. This reaction is catalysed by the enzyme nitrogenase (Nase). In N2 fixers associated with higher plants, only the molybdenum (Mo) dependent nitrogenase (Mo-Nase) has been identified. However, in many other N2 fixers two additional isoenzymes have been reported; the vanadium (V) dependent (V-Nase) and iron-only dependent nitrogenase (Fe-Nase). The role of these alternative nitrogenases (V-Nase and Fe-Nase) in natural habitats has been mostly overlooked, because they are found in communities that were not considered major contributors to N inputs. In recent years, N2 fixation associated with mosses and lichens has captured the interest of the scientific community for its importance toward global N input in high latitude ecosystems. Within this context, it is imperative to reconsider the role of alternative nitrogenases in these biomes. Here, I will present an overview of various findings, provided by different research groups, in the last two decade, suggesting that alternative nitrogenases could play an important role on N2 fixation in terrestrial ecosystems, especially in high latitude ones. I will discuss how these findings challenge the traditional view of Mo hegemony on N input in natural habitats and how it affects our conceptual models relating N2 fixation and trace metal dynamics in the environment. I will conclude by presenting my views on the importance to improve our understanding of the role of alternative nitrogenase in high latitude ecosystems; not only will this affect our fundamental understanding of N2 fixation and N cycling, it will also impact our ability to predict the response of these ecosystems to global climate change.

  12. Characterization of Diazotrophs Containing Mo-Independent Nitrogenases, Isolated from Diverse Natural Environments

    USDA-ARS?s Scientific Manuscript database

    Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other diazotrophic bacteria. Previously, we reported the isolation of seven diazotrophs with Molybdenum-independent nitrogenases from aquatic environments...

  13. Nitrogen isotope fractionation by alternative nitrogenases and past ocean anoxia

    PubMed Central

    Zhang, Xinning; Sigman, Daniel M.; Morel, François M. M.; Kraepiel, Anne M. L.

    2014-01-01

    Biological nitrogen fixation constitutes the main input of fixed nitrogen to Earth’s ecosystems, and its isotope effect is a key parameter in isotope-based interpretations of the N cycle. The nitrogen isotopic composition (δ15N) of newly fixed N is currently believed to be ∼–1‰, based on measurements of organic matter from diazotrophs using molybdenum (Mo)-nitrogenases. We show that the vanadium (V)- and iron (Fe)-only “alternative” nitrogenases produce fixed N with significantly lower δ15N (–6 to –7‰). An important contribution of alternative nitrogenases to N2 fixation provides a simple explanation for the anomalously low δ15N (<–2‰) in sediments from the Cretaceous Oceanic Anoxic Events and the Archean Eon. A significant role for the alternative nitrogenases over Mo-nitrogenase is also consistent with evidence of Mo scarcity during these geologic periods, suggesting an additional dimension to the coupling between the global cycles of trace elements and nitrogen. PMID:24639508

  14. Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

    PubMed

    Masukawa, Hajime; Inoue, Kazuhito; Sakurai, Hidehiro; Wolk, C Peter; Hausinger, Robert P

    2010-10-01

    Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.

  15. Maturation of nitrogenase cofactor—the role of a class E radical SAM methyltransferase NifB

    PubMed Central

    Hu, Yilin; Ribbe, Markus W.

    2016-01-01

    Nitrogenase catalyzes the important reactions of N2-, CO- and CO2-reduction at its active cofactor site. Designated the M-cluster, this complex metallocofactor is assembled through the generation of a characteristic 8Fe-core prior to the insertion of Mo and homocitrate that completes the stoichiometry of the M-cluster. NifB catalyzes the critical step of radical SAM-dependent carbide insertion that occurs concomitant with the insertion a “9th” sulfur and the rearrangement/coupling of two 4Fe-clusters into a complete 8Fe-core of the M-cluster. Further categorization of a family of NifB proteins as a new class of radical SAM methyltransferases suggests a general function of these proteins in complex metallocofactor assembly and provides a new platform for unveiling unprecedented chemical reactions catalyzed by biological systems. PMID:26969410

  16. The Inflammatory Response to Femoral Arterial Closure Devices: A Randomized Comparison Among FemoStop, AngioSeal, and Perclose

    SciTech Connect

    Jensen, Jens Saleh, Nawzad; Jensen, Ulf; Svane, Bertil; Joensson, Anders; Tornvall, Per

    2008-07-15

    The objectives of this study were to investigate whether the systemic inflammatory response differs, in patients undergoing coronary angiography, among the arterial closure devices FemoStop, AngioSeal, and Perclose. The study is a prospective and randomized study. We measured pre- and postprocedural C-reactive protein (CRP), fibrinogen, and interleukin-6 (IL-6) plasma levels and collected clinical and procedural data on 77 patients who underwent coronary angiography because of stable angina pectoris. Patients were randomized to the following device: FemoStop (mechanical compression), AngioSeal (anchor and collagen sponge), or Perclose (nonabsorbable suture). No patient group experienced an increased incidence of vascular complications. There were no differences among the three groups regarding CRP, fibrinogen, or IL-6 values before or after coronary angiography. IL-6 levels increased 6 h after the procedure in all groups (p < 0.01), however, the increase did not differ among the groups. After 30 days there were no increased values of CRP or fibrinogen. We conclude that the femoral arterial closure devices AngioSeal and Perclose do not enhance an inflammatory response after a diagnostic coronary angiography, measured by CRP, fibrinogen, and IL-6, compared to femoral arterial closure using a mechanical compression device.

  17. Ab initio study of energetics and magnetism of sigma phase in Co-Mo and Fe-Mo systems

    NASA Astrophysics Data System (ADS)

    Pavlů, J.; Vřešťál, J.; Šob, M.

    2016-02-01

    We analyse, from first-principles, the energetics and magnetic ordering of sigma phases in Co-Mo and Fe-Mo systems. Total energy differences between the sigma phase and Standard Element Reference (SER) structures are calculated in the whole concentration range at equilibrium volumes by means of the linear muffin-tin orbitals method in the atomic-sphere approximation (LMTO-ASA), the full-potential linearised augmented-plane waves (FLAPW) method and the pseudopotential approach. They are compared with the enthalpy of formation of sigma phase obtained from the phase equilibria calculations at higher temperature based on the semiempirical CALPHAD (CALculation of PHAse Diagram) method. It turns out that the binary sigma phases are more stable than the weighted average of the sigma phase of elemental constituents and that this stability for Fe-Mo is higher than for Co-Mo. On the other hand it was found that the binary sigma phases do not exhibit any stability with respect to the weighted average of the SER structures. The magnetic configurations in all systems are investigated and the stabilizing effect of magnetic order in sigma phase at 0 K is presented. It turns out that the atomic magnetic moment strongly depends on the type of occupied sublattice and total composition of the alloy.

  18. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    PubMed Central

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  19. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    PubMed

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  20. Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source

    SciTech Connect

    Ugalde, R.A.; Imperial, J.; Shah, V.K.; Brill, W.J.

    1985-12-01

    NifQ/sup -/ and Mol/sup -/ mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ/sup -/ and Mol/sup -/ mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. The data show that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. This study suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.

  1. Safety and efficacy of femoral artery closure with the FemoSeal(R) device after coronary angiography using a 7 French sheath.

    PubMed

    Wanitschek, M M; Suessenbacher, A; Dörler, J; Pachinger, O; Moes, N; Alber, H F

    2011-09-01

    Post-cardiac catheterization femoral artery hemostasis can be accomplished with several mechanisms, including the FemoSeal® hemostasis device which has been designed and approved for closure of 6 French (F) arterial puncture sites. The aim of this study was to investigate whether the FemoSeal® vascular closure device can effectively and safely seal 7F arterial puncture sites after diagnostic and interventional cardiac catheterizations. Femoral artery puncture sites of 50 consecutive patients undergoing cardiac catheterization were closed with the FemoSeal® vascular closure device, according to the manufacturer's instructions. Efficacy endpoints were time to hemostasis and successful ambulation. Safety endpoints included bleeding complications, vessel occlusion and pseudoaneurysms. Mean time to hemostasis was 57.8±26.3 seconds (0-125 seconds). Hemostasis was achieved in 100 percent of the 50 patients. One patient suffered minor bleeding the next day, i.e. local hematoma. This clinical study demonstrates that the FemoSeal® vascular closure device, initially approved for closure of 6F arterial puncture sites, shows promising efficacy and safety to seal a larger (7F) femoral arterial puncture sites after diagnostic and interventional cardiac catheterizations.

  2. Presence of a Vanadium Nitrogenase in Azotobacter paspali

    PubMed Central

    Fallik, Elazar; Hartel, Peter G.; Robson, Robert L.

    1993-01-01

    There have been no previous studies on the genetics of Azotobacter paspali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. paspali strains defective in the molybdenum nitrogenase so that alternative N2ases could be studied. The cosmid vector pTBE and genomic DNA fragments (∼50 kb) of A. paspali ATCC 23367 were used to construct a gene library in Escherichia coli. Recombinant cosmids containing sequences homologous to molybdenum nitrogenase nifDK structural genes were identified by hybridization. A 2.9-kb fragment bearing the putative nifDK genes of A. paspali was subcloned and mutagenized in vitro by the insertion of a kanamycin resistance gene cassette. The mutation was recombined into the chromosome of A. paspali with the suicide vector pCU101. One resultant mutant strain, AP2, was incapable of diazotrophic growth in a molybdenum-containing medium (Nif-) without vanadium but grew well in a molybdenum-deficient medium with vanadium. The nitrogenase system in AP2 reduced acetylene to ethylene and produced ethane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vanadium at levels up to 10 μM. These results are consistent with the existence of a vanadium nitrogenase system in A. paspali. PMID:16348965

  3. What Is the True Nitrogenase Reaction? A Guided Approach

    ERIC Educational Resources Information Center

    Ipata, Piero L.; Pesi, Rossana

    2015-01-01

    Only diazotrophic bacteria, called "Rizhobia," living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N[subscript 2]. In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an…

  4. Posttranslational regulatory system for nitrogenase activity in Azospirillum spp.

    PubMed Central

    Fu, H A; Hartmann, A; Lowery, R G; Fitzmaurice, W P; Roberts, G P; Burris, R H

    1989-01-01

    The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum. Images PMID:2504694

  5. What Is the True Nitrogenase Reaction? A Guided Approach

    ERIC Educational Resources Information Center

    Ipata, Piero L.; Pesi, Rossana

    2015-01-01

    Only diazotrophic bacteria, called "Rizhobia," living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N[subscript 2]. In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an…

  6. Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement

    PubMed Central

    Spatzal, Thomas; Schlesier, Julia; Burger, Eva-Maria; Sippel, Daniel; Zhang, Limei; Andrade, Susana L.A.; Rees, Douglas C.; Einsle, Oliver

    2016-01-01

    The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S=3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the recently revised oxidation state assignment for the molybdenum ion, providing the first spatially resolved picture of the resting-state electron distribution within FeMoco. This might provide the long-sought experimental basis for a generally accepted theoretical description of the cluster that is in line with available spectroscopic and functional data. PMID:26973151

  7. Cofactor engineering for advancing chemical biotechnology.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.

  8. Comparative electronic structures of nitrogenase FeMoco and FeVco† †Electronic supplementary information (ESI) available: Additional figures and tables, computational data and information. See DOI: 10.1039/c7dt00128b Click here for additional data file.

    PubMed Central

    Rees, Julian A.; Bjornsson, Ragnar; Kowalska, Joanna K.; Lima, Frederico A.; Schlesier, Julia; Sippel, Daniel; Weyhermüller, Thomas

    2017-01-01

    An investigation of the active site cofactors of the molybdenum and vanadium nitrogenases (FeMoco and FeVco) was performed using high-resolution X-ray spectroscopy. Synthetic heterometallic iron–sulfur cluster models and density functional theory calculations complement the study of the MoFe and VFe holoproteins using both non-resonant and resonant X-ray emission spectroscopy. Spectroscopic data show the presence of direct iron–heterometal bonds, which are found to be weaker in FeVco. Furthermore, the interstitial carbide is found to perturb the electronic structures of the cofactors through highly covalent Fe–C bonding. The implications of these conclusions are discussed in light of the differential reactivity of the molybdenum and vanadium nitrogenases towards various substrates. Possible functional roles for both the heterometal and the interstitial carbide are detailed. PMID:28154874

  9. Molybdenum cofactor and human disease.

    PubMed

    Schwarz, Guenter

    2016-04-01

    Four molybdenum-dependent enzymes are known in humans, each harboring a pterin-based molybdenum cofactor (Moco) in the active site. They catalyze redox reactions using water as oxygen acceptor or donator. Moco is synthesized by a conserved biosynthetic pathway. Moco deficiency results in a severe inborn error of metabolism causing often early childhood death. Disease-causing symptoms mainly go back to the lack of sulfite oxidase (SO) activity, an enzyme in cysteine catabolism. Besides their name-giving functions, Mo-enzymes have been recognized to catalyze novel reactions, including the reduction of nitrite to nitric oxide. In this review we cover the biosynthesis of Moco, key features of Moco-enzymes and focus on their deficiency. Underlying disease mechanisms as well as treatment options will be discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

    George, Simon J; Hernandez, Jose A; Jimenez-Vicente, Emilio; Echavarri-Erasun, Carlos; Rubio, Luis M

    2016-09-27

    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.

  11. The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria

    PubMed Central

    Yokoyama, Kenichi; Leimkühler, Silke

    2016-01-01

    Molybdenum is the only second row transition metal essential for biological systems, which is biologically available as molybdate ion. In eukarya, bacteria and archaea, molybdenum is bound to either to a tricyclic pyranopterin, thereby forming the molybdenum cofactor (Moco), or in some bacteria to the FeS cluster based iron-molybdenum cofactor (FeMoco), which forms the active site of nitrogenase. To date more than 50 Moco-containing enzymes have been purified and biochemically or structurally characterized. The physiological role of molybdenum in these enzymes is fundamental to organisms, since the reactions include the catalysis of key steps in carbon, nitrogen and sulfur metabolism. The catalyzed reactions are in most cases oxo-transfer reactions or the hydroxylation of carbon centers. The biosynthesis of Moco has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the biosynthesis and maturation of molybdoenzymes and the biosynthesis and distribution of FeS clusters has been identified in the last years: 1) The synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) The sulfurtransferase for the dithiolene group in Moco is common also for the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the modification of the active site with a sulfur atom additionally involves a sulfurtransferase, 4) most molybdoenzymes in bacteria require FeS clusters as additional redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. PMID:25268953

  12. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria.

    PubMed

    McKinlay, James B; Harwood, Caroline S

    2010-06-29

    The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO(2). A less appreciated function of CO(2) fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO(2) fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with (13)C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO(2) and then fixed 68% of this CO(2) into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO(2) fixation plays a major role in cofactor recycling. When H(2) production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO(2) was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N(2) fixation and CO(2) fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO(2) fixation to recycle reduced cofactors deserves closer scrutiny.

  13. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria

    PubMed Central

    McKinlay, James B.; Harwood, Caroline S.

    2010-01-01

    The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO2. A less appreciated function of CO2 fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO2 fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with 13C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO2 and then fixed 68% of this CO2 into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO2 fixation plays a major role in cofactor recycling. When H2 production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO2 was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N2 fixation and CO2 fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO2 fixation to recycle reduced cofactors deserves closer scrutiny. PMID:20558750

  14. Engineering redox balance through cofactor systems.

    PubMed

    Chen, Xiulai; Li, Shubo; Liu, Liming

    2014-06-01

    Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Mechanistic insights into nitrogen fixation by nitrogenase enzymes.

    PubMed

    Varley, J B; Wang, Y; Chan, K; Studt, F; Nørskov, J K

    2015-11-28

    Biological nitrogen fixation by nitrogenase enzymes is a process that activates dinitrogen (N2) one of the most inert molecules in nature, within the confines of a living organism and at ambient conditions. Despite decades of study, there are still no complete explanations as to how this is possible. Here we describe a model of N2 reduction using the Mo-containing nitrogenase (FeMoco) that can explain the reactivity of the active site via a series of electrochemical steps that reversibly unseal a highly reactive Fe edge site. Our model can explain the 8 proton-electron transfers involved in biological ammonia synthesis within the kinetic scheme of Lowe and Thorneley, the obligatory formation of one H2 per N2 reduced, and the behavior of known inhibitors.

  16. Physiology of Ex Planta Nitrogenase Activity in Rhizobium japonicum†

    PubMed Central

    Agarwal, Arun K.; Keister, Donald L.

    1983-01-01

    Thirty-nine wild-type strains of Rhizobium japonicum have been studied for their ability to synthesize nitrogenase ex planta in defined liquid media under microaerobic conditions. Twenty-one produced more than trace amounts of acetylene reduction activity, but only a few of these yielded high activity. The oxygen response curves were similar for most of the nitrogenase-positive strains. The strains derepressible for activity had several phenotypic characteristics different from non-derepressible strains. These included slower growth and lower oxygen consumption under microaerobic conditions and lower extracellular polysaccharide production. Extracellular polysaccharide production during growth on gluconate in every nitrogenase-positive strain assayed was lower under both aerobic and microaerobic conditions than the non-derepressible strains. These phenotypic characteristics may be representative of a genotype of a subspecies of R. japonicum. These studies were done in part to enlarge the base number of strains available for studies on the physiology, biochemistry, and genetics of nitrogen fixation. PMID:16346295

  17. Physiology of ex planta nitrogenase activity in Rhizobium japonicum

    SciTech Connect

    Agarwal, A.K.; Keister, D.L.

    1983-05-01

    Thirty-nine wild-type strains of Rhizobium japonicum have been studied for their ability to synthesize nitrogenase ex planta in defined liquid media under microaerobic conditions. Twenty-one produced more than trace amounts of acetylene reduction activity, but only a few of these yielded high activity. The oxygen response curves were similar for most of the nitrogenase-positive strains. The strains derepressible for activity had several phenotypic characteristics different from non-derepressible strains. These included slower growth and lower oxygen consumption under microaerobic conditions and lower extracellular polysaccharide production. Extracellular polysaccharide production during growth on gluconate in every nitrogenase-positive strain assayed was lower under both aerobic and microaerobic conditions than the non-depressible strains. These phenotypic characteristics may be representative of a genotype of a subspecies of R. japonicum. These studies were done in part to enlarge the base number of strains available for studies on the physiology, biochemistry, and genetics of nitrogen fixation. (35 Refs.)

  18. Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions

    PubMed Central

    Esteves-Ferreira, Alberto A.; Cavalcanti, João Henrique Frota; Vaz, Marcelo Gomes Marçal Vieira; Alvarenga, Luna V.; Nunes-Nesi, Adriano; Araújo, Wagner L.

    2017-01-01

    Abstract Cyanobacteria is a remarkable group of prokaryotic photosynthetic microorganisms, with several genera capable of fixing atmospheric nitrogen (N2) and presenting a wide range of morphologies. Although the nitrogenase complex is not present in all cyanobacterial taxa, it is spread across several cyanobacterial strains. The nitrogenase complex has also a high theoretical potential for biofuel production, since H2 is a by-product produced during N2 fixation. In this review we discuss the significance of a relatively wide variety of cell morphologies and metabolic strategies that allow spatial and temporal separation of N2 fixation from photosynthesis in cyanobacteria. Phylogenetic reconstructions based on 16S rRNA and nifD gene sequences shed light on the evolutionary history of the two genes. Our results demonstrated that (i) sequences of genes involved in nitrogen fixation (nifD) from several morphologically distinct strains of cyanobacteria are grouped in similarity with their morphology classification and phylogeny, and (ii) nifD genes from heterocytous strains share a common ancestor. By using this data we also discuss the evolutionary importance of processes such as horizontal gene transfer and genetic duplication for nitrogenase evolution and diversification. Finally, we discuss the importance of H2 synthesis in cyanobacteria, as well as strategies and challenges to improve cyanobacterial H2 production. PMID:28323299

  19. Isotopic Biomarkers of Nitrogenase Metalloenzymes: Forging Links Between the Cycles of Nitrogen and Trace Metals

    NASA Astrophysics Data System (ADS)

    Zhang, X.; McRose, D. L.; Darnajoux, R.; Bellenger, J. P.; Kraepiel, A. M. L.

    2015-12-01

    Biological N2 fixation, catalyzed by the metalloenzyme nitrogenase, is a critical process that makes life possible on Earth. Environmental N2 fixation has been automatically attributed to canonical Mo-based nitrogenases despite over two decades of knowledge that two other metalloenzyme forms of nitrogenase exist: those containing catalytic V or Fe-only. A key area of missing information is the contribution of the "alternative" V and Fe-only nitrogenases, as the interpretation of field data to construct budgets and assess N availability depends on the type of nitrogenase metalloenzyme used to fix N2. Additionally, substantial changes in metal speciation over geological time may have favored the use of different metalloenzymes, with implications for evolution of the biosphere. Despite the potential importance of alternative nitrogenases in modern and ancient N cycling, few methods can determine their contributions to environmental N2 fixation. Here, we present new isotopic methods to distinguish between the activities of Mo, V, and Fe-only nitrogenases. We show evidence for alternative N2 fixation in diverse environments (cyanolichens, microbial mats, sediments, leaf litter), thereby linking a key process in the nitrogen cycle to specific metalloenzyme forms of nitrogenase. The results invite a reexamination of the conditions under which the different nitrogenase metalloenzymes are active and may lead to new insights into the coupling of the cycles of nitrogen and trace metals.

  20. High resistive nanocrystalline Fe-M-O (M=Hf, Zr, rare-earth metals) soft magnetic films for high-frequency applications (invited)

    NASA Astrophysics Data System (ADS)

    Hayakawa, Y.; Makino, A.; Fujimori, H.; Inoue, A.

    1997-04-01

    Microstructure, soft magnetic properties, and applications of high resistive Fe-M-O (M=Hf, Zr, rare-earth metals) were studied. The Fe-M-O films are composed of bcc nanograins and amorphous phases with larger amounts of M and O elements which chemically combine each other. Consequently, the amorphous phases have high electrical resistivity. The compositional dependence of magnetic properties, electrical resistivity, and structure have been almost clarified. For example, the high magnetization of 1.3 T, high permeability of 1400 at 100 MHz and the high electrical resistivity of 4.1 μΩ m are simultaneously obtained for as-deposited Fe62Hf11O27 nanostructured film fabricated by rf reactive sputtering in a static magnetic field. Furthermore, Co addition to Fe-M-O films improves the frequency characteristics mainly by the increase in the crystalline anisotropy of the nanograins. The Co44.3Fe19.1Hf14.5O22.1 film exhibits the quality factor (Q=μ'/μ'') of 61 and the μ' of 170 at 100 MHz as well as the high Is of 1.1 T. This frequency characteristics is considered to be superior to the other films already reported. The films also exhibit high corrosion resistance in an isotonic sodium chloride solution. Therefore, these films enable us to realize the high-frequency magnetic devices, such as thin-film inductors and transformers for microswitching converters and ultrahigh-density recording heads.

  1. Inhibition of chymotrypsin by heparin cofactor II.

    PubMed Central

    Church, F C; Noyes, C M; Griffith, M J

    1985-01-01

    Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin. PMID:3863104

  2. Catalytic formation of a nitrogenase iron-sulfur cluster.

    PubMed

    Zheng, L; Dean, D R

    1994-07-22

    Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme comprised of two component proteins called the Fe protein and the MoFe protein. Both nitrogenase component proteins contain metalloclusters. The Azotobacter vinelandii nifS gene product (NifS), which is required for full activation of the nitrogenase component proteins, is a pyridoxal phosphate enzyme and is able to catalyze the desulfurization of L-cysteine to yield sulfur and L-alanine (Zheng, L., White, R. H., Cash, V.L., Jack, R.F., and Dean, D.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758). An enzyme-bound persulfide that was identified as an intermediate in the cysteine desulfurization reaction catalyzed by NifS has been suggested as a possible S-donor in formation of the iron-sulfide cores of the nitrogenase metalloclusters. In the present work it is shown that NifS is able to effectively catalyze activation of an apo-form of the Fe protein that was prepared by removal of its Fe4S4 cluster using the chelator, alpha,alpha'-dipyridyl. The reconstitution reaction includes apo-Fe protein, NifS, L-cysteine, ferrous ion, dithiothreitol, and MgATP. Reconstitution of the inactive apo-Fe protein catalyzed by NifS results in 80-95% recovery of the original activity and yields an Fe protein having the normal electron paramagnetic resonance spectra properties associated with the Fe protein's Fe4S4 cluster. An altered NifS protein, NifS-Ala325, which lacks the desulfurase activity and is unable to from the NifS-bound persulfide, is not able to catalyze reactivation of the apo-Fe protein. These in vitro results support the proposal that NifS activity provides the inorganic sulfide necessary for in vivo formation of the nitrogenase metalloclusters. Moreover, because NifS has recently been shown to be a member of a highly homologous gene family, it appears that pyridoxal phosphate chemistry might play a general role in iron-sulfur cluster assembly.

  3. The mechanistically significant coordination chemistry of dinitrogen at FeMo-co, the catalytic site of nitrogenase.

    PubMed

    Dance, Ian

    2007-02-07

    Reported here is a comprehensive theoretical investigation of the binding of N(2) to the Fe(7)MoS(9)N(homocitrate)(cysteine)(histidine) active site (FeMo-co) of the enzyme nitrogenase, as a prerequisite to elucidation of the chemical mechanism of the catalyzed reduction to NH(3). The degree and type of hydrogenation of FeMo-co, with H atoms and possibly an H(2) molecule, are key variables, following the Thorneley-Lowe kinetic scheme. Ninety-four local energy minima were located for N(2) coordinated in eta(2) (side) and eta(1) (end) modes at the endo and exo coordination positions of Fe2 and Fe6. The stabilities of 57 representative structures are assessed by calculation of the reaction profiles and activation energies for the association and dissociation of N(2). Barriers to association of N(2) depend mainly on the location of the hydrogenation and the location of N(2) coordination, while dissociation barriers depend primarily on whether N(2) is eta(2)- and eta(1)-coordinated, and secondarily on the location of the hydrogenation. Increased negative charge on FeMo-co increases the barriers, while C in place of N at the center of FeMo-co has little effect. The interactions of the models of ligated FeMo-co with the surrounding protein, including proteins with mutations of key amino acids, are assessed by in silico cofactor transplantations and calculations of protein strain energies. From these results, which identify models involving contacts and interactions with the surrounding residues that have been shown by mutation to affect the N(2) activity of nitrogenase, and from the N(2) coordination profiles, it is concluded that endo-eta(1)-N(2) coordination at Fe6 is most probable. There is strong reason to believe that the mechanism of nitrogenase will involve one or more of the preferred models presented here, and a detailed foundation of structures and principles is now available for postulation and calculation of the profiles of the steps in which H atoms bound to

  4. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  5. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  6. The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

    DOE PAGES

    Ledbetter, Rhesa N.; Garcia Costas, Amaya M.; Lubner, Carolyn E.; ...

    2017-07-13

    The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxinmore » semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered ..delta..rnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Altogether, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.« less

  7. The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

    PubMed

    Ledbetter, Rhesa N; Garcia Costas, Amaya M; Lubner, Carolyn E; Mulder, David W; Tokmina-Lukaszewska, Monika; Artz, Jacob H; Patterson, Angela; Magnuson, Timothy S; Jay, Zackary J; Duan, H Diessel; Miller, Jacquelyn; Plunkett, Mary H; Hoben, John P; Barney, Brett M; Carlson, Ross P; Miller, Anne-Frances; Bothner, Brian; King, Paul W; Peters, John W; Seefeldt, Lance C

    2017-08-15

    The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

  8. Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency.

    PubMed

    Tejada-Jiménez, Manuel; Gil-Díez, Patricia; León-Mediavilla, Javier; Wen, Jiangqi; Mysore, Kirankumar S; Imperial, Juan; González-Guerrero, Manuel

    2017-08-14

    Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotiana tabacum (Tnt1) insertional mutant line were carried out in the one M. truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase. © 2017 The Authors New Phytologist © 2017 New Phytologist Trust.

  9. Overproduction of nitrogenase by nitrogen-limited cultures of Rhodopseudomonas palustris.

    PubMed Central

    Arp, D J; Zumft, W G

    1983-01-01

    Rhodopseudomonas palustris cells grown on limiting nitrogen produced four- to eightfold higher nitrogenase specific activity relative to cells sparged with N2. The high activity of N-limited cells was the result of overproduction of the nitrogenase proteins. This was shown by four independent techniques: (i) titration of the Mo-Fe protein in cell-free extracts with Fe protein from Azotobacter vinelandii; (ii) direct detection of the subunits of Mo-Fe protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (iii) monitoring of the electron paramagnetic resonance spectrum of Mo-Fe protein in whole cells; and (iv) immunological assay of the Fe protein level with an antiserum against the homologous protein of Rhodospirillum rubrum. The derepressed level of nitrogenase found in N2-grown cells was not due to an increased turnover of nitrogenase. The apparent half-lives of nitrogenase in N2-grown and N-limited cells were 58 and 98 h, respectively, but were too long to account for the difference in enzyme level. Half-lives were determined by measuring nitrogenase after repression of de novo synthesis by ammonia and subsequent release of nitrogenase switch-off by methionine sulfoximine. Observations were extended to R. rubrum, Rhodopseudomonas capsulata, and Rhodomicrobium vannielii and indicated that overproduction of nitrogenase under nitrogen limitation is not an exceptional property of R. palustris, but rather a general property of phototrophic bacteria. Images PMID:6402491

  10. Influence of External Nitrogen on Nitrogenase Enzyme Activity and Auxin Production in Herbaspirillum seropedicae (Z78).

    PubMed

    Yin, Tan Tzy; Pin, Ui Li; Ghazali, Amir Hamzah Ahmad

    2015-04-01

    The production of nitrogenase enzyme and auxins by free living diazotrophs has the potential to influence the growth of host plants. In this study, diazotrophs were grown in the presence of various concentrations of nitogen (N) to determine the optimal concentration of N for microbial growth stimulation, promotion of gaseous N (N2) fixation, and phytohormone production. Therefore, we investigate whether different levels of N supplied to Herbaspirillum seropedicae (Z78) have significant effects on nitrogenase activity and auxin production. The highest nitrogenase activity and the lowest auxin production of H. seropedicae (Z78) were both recorded at 0 gL(-1) of NH4Cl. Higher levels of external N caused a significant decrease in the nitrogenase activity and an increased production of auxins. In a subsequent test, two different inoculum sizes of Z78 (10(6) and 10(12) cfu/ml) were used to study the effect of different percentages of acetylene on nitrogenase activity of the inoculum via the acetylene reduction assay (ARA). The results showed that the optimal amount of acetylene required for nitrogenase enzyme activity was 5% for the 10(6) cfu/ml inoculum, whereas the higher inoculum size (10(12) cfu/ml) required at least 10% of acetylene for optimal nitrogenase activity. These findings provide a clearer understanding of the effects of N levels on diazotrophic nitrogenase activity and auxin production, which are important factors influencing plant growth.

  11. Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

    SciTech Connect

    Danyal, Karamatullah; Shaw, Sudipta; Page, Taylor R.; Duval, Simon; Horitani, Masaki; Marts, Amy R.; Lukoyanov, Dmitriy; Dean, Dennis R.; Raugei, Simone; Hoffman, Brian M.; Seefeldt, Lance C.; Antony, Edwin

    2016-10-04

    Mo-dependent nitrogenase catalyzes the biological reduction of atmospheric dinitrogen (N2) to two ammonia (NH3) molecules, through the action of two component proteins, the MoFe protein and the Fe protein. The catalytic MoFe protein is a symmetric dimer of αβ units, each of which contains one active site FeMo-co (FeMo-co; [7Fe-9S-Mo-C-homocitrate]) and an electron-carrier P cluster. Each half of the nitrogenase ternary complex, in which one Fe protein with two bound ATP molecules has bound to each MoFe protein αβ unit, undergoes an electron transfer (ET) cycle with ET from a Fe protein [4Fe-4S] cluster into its αβ unit followed by the hydrolysis of the two ATP to two ADP and two Pi. The prevailing model holds that each αβ unit of the MoFe protein functions independently. We now report that the ET cycle exhibits negative cooperativity, with ET and ATP hydrolysis in one half of the ternary nitrogenase complex suppressing these processes in the other half. The observed ET, ATP hydrolysis, and Pi release behavior is captured in a global fit to a two-branch negative-cooperativity kinetic model. A possible mechanism for communication between the two halves of MoFe protein is suggested by normal mode analysis showing correlated and anti-correlated motions between the two nitrogenase αβ halves. EPR spectra furthermore show small differences between those of resting-state and singly-reduced MoFe protein that can be attributed to an intra-complex allosteric perturbation of the resting-state FeMo-co in one αβ unit by reduction of FeMo-co in the other. This work is supported as a part of the Biological and Electron Transfer and Catalysis (EFRC) program, an Energy Frontiers Research Center funded by the US Department of Energy (DOE), Office of Science (DE-SC0012518) to LCS, by National Institutes of Health (NIH) grants HL 63203 and GM 111097to BMH, and R15GM110671 to EA, and by the Division of Chemical Sciences, Geosciences, and Bio-Sciences, DOE to SR. The protein

  12. Cofactor engineering for more efficient production of chemicals and biofuels.

    PubMed

    Wang, Meng; Chen, Biqiang; Fang, Yunming; Tan, Tianwei

    2017-09-20

    Cofactors are involved in numerous intracellular reactions and critically influence redox balance and cellular metabolism. Cofactor engineering can support and promote the biocatalysis process, even help driving thermodynamically unfavorable reactions forwards. To achieve efficient production of chemicals and biofuels, cofactor engineering strategies such as altering cofactor supply or modifying reactants' cofactor preference have been developed to maintain redox balance. This review focuses primarily on the effects of cofactor engineering on carbon and energy metabolism. Coupling carbon metabolism with cofactor engineering can promote large-scale production, and even offer possibilities for producing new products or converting new materials. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Electron inventory, kinetic assignment (E(n)), structure, and bonding of nitrogenase turnover intermediates with C2H2 and CO.

    PubMed

    Lee, Hong-In; Sørlie, Morten; Christiansen, Jason; Yang, Tran-Chin; Shao, Junlong; Dean, Dennis R; Hales, Brian J; Hoffman, Brian M

    2005-11-16

    Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.

  14. High resistive nanocrystalline Fe-M-O (M=Hf, Zr, rare-earth metals) soft magnetic films for high-frequency applications (invited)

    SciTech Connect

    Hayakawa, Y.; Makino, A.; Fujimori, H.; Inoue, A.

    1997-04-01

    Microstructure, soft magnetic properties, and applications of high resistive Fe-M-O (M=Hf, Zr, rare-earth metals) were studied. The Fe-M-O films are composed of bcc nanograins and amorphous phases with larger amounts of M and O elements which chemically combine each other. Consequently, the amorphous phases have high electrical resistivity. The compositional dependence of magnetic properties, electrical resistivity, and structure have been almost clarified. For example, the high magnetization of 1.3 T, high permeability of 1400 at 100 MHz and the high electrical resistivity of 4.1 {mu}{Omega}m are simultaneously obtained for as-deposited Fe{sub 62}Hf{sub 11}O{sub 27} nanostructured film fabricated by rf reactive sputtering in a static magnetic field. Furthermore, Co addition to Fe-M-O films improves the frequency characteristics mainly by the increase in the crystalline anisotropy of the nanograins. The Co{sub 44.3}Fe{sub 19.1}Hf{sub 14.5}O{sub 22.1} film exhibits the quality factor (Q={mu}{sup {prime}}/{mu}{sup {prime}{prime}}) of 61 and the {mu}{sup {prime}} of 170 at 100 MHz as well as the high Is of 1.1 T. This frequency characteristics is considered to be superior to the other films already reported. The films also exhibit high corrosion resistance in an isotonic sodium chloride solution. Therefore, these films enable us to realize the high-frequency magnetic devices, such as thin-film inductors and transformers for microswitching converters and ultrahigh-density recording heads. {copyright} {ital 1997 American Institute of Physics.}

  15. Enhanced Hydrogen Production by Co-cultures of Hydrogenase and Nitrogenase in Escherichia coli.

    PubMed

    Lee, Hyun Jeong; Sekhon, Simranjeet Singh; Kim, Young Su; Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2016-03-01

    Rhodobacter sphaeroides is a bacterium that can produce hydrogen by interaction with hydrogenase and nitrogenase. We report a hydrogen production system using co-cultivation of hydrogenase in liquid medium and immobilized nitrogenase in Escherichia coli. The recombinant plasmid has been constructed to analyze the effect of hydrogen production on the expression of hupSL hydrogenase and nifHDK nitrogenase isolated from R. sphaeroides. All recombinant E. coli strains were cultured anaerobically, and cells for nitrogenase were immobilized in agar gel, whereas cells for hydrogenase were supplemented on the nitrogenase agar gel. The hupSL hydrogenase has been observed to enhance hydrogen production and hydrogenase activity under co-culture with nifHDK nitrogenase. The maximum hydrogen production has been obtained at an agar gel concentration and a cell concentration for co-culture of 2 % and 6.4 × 10(8) CFU. Thus, co-culture of hupSL hydrogenase and nifHDK nitrogenase provides a promising route for enhancing the hydrogen production and hydrogenase activity.

  16. Complementary functioning of the component proteins of nitrogenase from several bacteria.

    PubMed Central

    Emerich, D W; Burris, R H

    1978-01-01

    The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity. PMID:659370

  17. Magnetostructural coupling behavior at the ferromagnetic transition in double-perovskite S r2FeMo O6

    NASA Astrophysics Data System (ADS)

    Yang, Dexin; Harrison, Richard J.; Schiemer, Jason A.; Lampronti, Giulio I.; Liu, Xueyin; Zhang, Fenghua; Ding, Hao; Liu, Yan'gai; Carpenter, Michael A.

    2016-01-01

    The ordered double-perovskite S r2FeMo O6 (SFMO) possesses remarkable room-temperature low-field colossal magnetoresistivity and transport properties which are related, at least in part, to combined structural and magnetic instabilities that are responsible for a cubic-tetragonal phase transition near 420 K. A formal strain analysis combined with measurements of elastic properties from resonant ultrasound spectroscopy reveal a system with weak biquadratic coupling between two order parameters belonging to Γ4+ and m Γ4+ of parent space group F m 3 ¯m . The observed softening of the shear modulus by ˜50% is due to the classical effects of strain/order parameter coupling at an improper ferroelastic (Γ4+) transition which is second order in character, while the ferromagnetic order parameter (m Γ4+ ) couples only with volume strain. The influence of a third order parameter, for ordering of Fe and Mo on crystallographic B sites, is to change the strength of coupling between the Γ4+ order parameter and the tetragonal shear strain due to the influence of changes in local strain heterogeneity at a unit cell scale. High anelastic loss below the transition point reveals the presence of mobile ferroelastic twin walls which become pinned by oxygen vacancies in a temperature interval near 340 K. The twin walls must be both ferroelastic and ferromagnetic, but due to the weak coupling between the magnetic and structural order parameters it should be possible to pull them apart with a weak magnetic field. These insights into the role of strain coupling and relaxational effects in a system with only weak coupling between three order parameters allow rationalization and prediction of how static and dynamic properties of the material might be tuned in thin film form by choice of strain contrast with a substrate.

  18. Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.

    PubMed Central

    Hallenbeck, P C; Meyer, C M; Vignais, P M

    1982-01-01

    Nitrogenase proteins were isolated from cultures of the photosynthetic bacterium Rhodopseudomonas capsulata grown on a limiting amount of ammonia. Under these conditions, the nitrogenase N2ase A was active in vivo, and nitrogenase activity in vitro was not dependent upon manganese and the activating factor. The nitrogenase proteins were also isolated from nitrogen-limited cultures in which the in vivo nitrogenase activity had been stopped by an ammonia shock. This nitrogenase activity, N2ase R, showed an in vitro requirement for manganese and the activating factor for maximal activity. The Mo-Fe protein (dinitrogenase) was composed of two dissimilar subunits with molecular weights of 55,000 and 59,500; the Fe protein (dinitrogenase reductase), from either type of culture, was composed of a single subunit (molecular weight), 33,500). The metal and acid labile sulfur contents of both nitrogenase proteins were similar to those found for previously isolated nitrogenases. The Fe proteins from both N2ase A and N2ase R contained phosphate and ribose, 2 mol of each per mol of N2ase R Fe protein and about 1 mol of each per mol of N2ase A Fe protein. The greatest difference between the two types of Fe protein was that the N2ase R Fe protein contained about 1 mol per mol of an adenine-like molecule, whereas the N2ase A Fe protein content of this compound was insignificant. These results are compared with various models previously presented for the short-term regulation of nitrogenase activity in the photosynthetic bacteria. PMID:6799495

  19. Regulation of Three Nitrogenase Gene Clusters in the Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Thiel, Teresa; Pratte, Brenda S.

    2014-01-01

    The filamentous cyanobacterium Anabaena variabilis ATCC 29413 fixes nitrogen under aerobic conditions in specialized cells called heterocysts that form in response to an environmental deficiency in combined nitrogen. Nitrogen fixation is mediated by the enzyme nitrogenase, which is very sensitive to oxygen. Heterocysts are microxic cells that allow nitrogenase to function in a filament comprised primarily of vegetative cells that produce oxygen by photosynthesis. A. variabilis is unique among well-characterized cyanobacteria in that it has three nitrogenase gene clusters that encode different nitrogenases, which function under different environmental conditions. The nif1 genes encode a Mo-nitrogenase that functions only in heterocysts, even in filaments grown anaerobically. The nif2 genes encode a different Mo-nitrogenase that functions in vegetative cells, but only in filaments grown under anoxic conditions. An alternative V-nitrogenase is encoded by vnf genes that are expressed only in heterocysts in an environment that is deficient in Mo. Thus, these three nitrogenases are expressed differentially in response to environmental conditions. The entire nif1 gene cluster, comprising at least 15 genes, is primarily under the control of the promoter for the first gene, nifB1. Transcriptional control of many of the downstream nif1 genes occurs by a combination of weak promoters within the coding regions of some downstream genes and by RNA processing, which is associated with increased transcript stability. The vnf genes show a similar pattern of transcriptional and post-transcriptional control of expression suggesting that the complex pattern of regulation of the nif1 cluster is conserved in other cyanobacterial nitrogenase gene clusters. PMID:25513762

  20. A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase.

    PubMed

    Simpson, F B; Burris, R H

    1984-06-08

    The effect of a partial pressure of nitrogen of 50 atmospheres (5065 kilopascals ) on the hydrogen evolution reaction of nitrogenase has been investigated. Evolution of hydrogen was not blocked completely by 50 atmospheres of nitrogen in any of four experiments; rather, 27.3 +/- 2.4 percent of the total electron flux through nitrogenase was directed toward production of hydrogen. The ratio of hydrogen evolved to nitrogen fixed was close to 1:1, which implies that hydrogen evolution is obligatory in the fixation of molecular nitrogen by nitrogenase.

  1. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  2. Nitrogenase structure and function relationships by density functional theory.

    PubMed

    Harris, Travis V; Szilagyi, Robert K

    2011-01-01

    Modern density functional theory has tremendous potential with matching popularity in metalloenzymology to reveal the unseen atomic and molecular details of structural data, spectroscopic measurements, and biochemical experiments by providing insights into unobservable structures and states, while also offering theoretical justifications for observed trends and differences. An often untapped potential of this theoretical approach is to bring together diverse experimental structural and reactivity information and allow for these to be critically evaluated at the same level. This is particularly applicable for the tantalizingly complex problem of the structure and molecular mechanism of biological nitrogen fixation. In this chapter we provide a review with extensive practical details of the compilation and evaluation of experimental data for an unbiased and systematic density functional theory analysis that can lead to remarkable new insights about the structure-function relationships of the iron-sulfur clusters of nitrogenase.

  3. Negative cooperativity in the nitrogenase Fe protein electron delivery cycle

    PubMed Central

    Danyal, Karamatullah; Shaw, Sudipta; Page, Taylor R.; Duval, Simon; Horitani, Masaki; Marts, Amy R.; Lukoyanov, Dmitriy; Dean, Dennis R.; Raugei, Simone; Hoffman, Brian M.; Seefeldt, Lance C.; Antony, Edwin

    2016-01-01

    Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves. PMID:27698129

  4. Improvement of the photocatalytic properties of TiO2 by (Fe+Mo) co-doping—A possible way to retard the recombination process

    NASA Astrophysics Data System (ADS)

    Guo, Junbo; Gan, Zhanghua; Lu, Zhihong; Liu, Jing; Xi, Jingjing; Wan, Yang; Le, Lin; Liu, Hailin; Shi, Jing; Xiong, Rui

    2013-09-01

    Low visible light absorption and high charge carrier recombination rate are two main disadvantages of TiO2 as a photocatalyst which severely limit its practical applications. To overcome the problems, Fe mono-doped and (Fe+Mo) co-doped TiO2 were synthesized and studied. It was found that (Fe+Mo) co-doping can further increase the visible absorption and improve the photocatalytic property of TiO2 compared with Fe mono-doping; Fe mono-doping improves the photocatalytic property of TiO2 only at very low doping level (Fe concentration less than 1.0%), while by co-doping a small amount of Mo with Fe, the effective doping concentration of Fe can be pushed to a higher level and the photocatalytic property of TiO2 can be further improved. Photoluminescence spectra indicated that Mo dopant may play a role in retarding the recombination process when co-doped into TiO2 with Fe. The mechanism behind was discussed. It was suggested that doping a small amount of Mo into Fe-TiO2 might be an efficient way to further improve the photocatalytic property of Fe-TiO2 without losing its photocatalytic specificity.

  5. Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

    PubMed Central

    Hartmann, A; Burris, R H

    1987-01-01

    The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum. Images PMID:2880836

  6. Soil surface disturbances in cold deserts: Effects on nitrogenase activity in cyanobacterial-lichen soil crusts

    USGS Publications Warehouse

    Belnap, Jayne

    1996-01-01

    CyanobacteriaMichen soil crusts can be a dominant source of nitrogen for cold-desert ecosystems. Effects of surface disturbance from footprints, bike and vehicle tracks on the nitrogenase activity in these crusts was investigated. Surface disturbances reduced nitrogenase activity by 30-100%. Crusts dominated by the cyanobacterium Microcoleus vaginatus on sandy soils were the most susceptible to disruption; crusts on gypsiferous soils were the least susceptible. Crusts where the soil lichen Collema tenax was present showed less immediate effects; however, nitrogenase activity still declined over time. Levels of nitrogenase activity reduction were affected by the degree of soil disruption and whether sites were dominated by cyanobacteria with or without heterocysts. Consequently, anthropogenic surface disturbances may have serious implications for nitrogen budgets in these ecosystems.

  7. Oxygen and the light-dark cycle of nitrogenase activity in two unicellular cyanobacteria.

    PubMed

    Compaoré, Justine; Stal, Lucas J

    2010-01-01

    Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well-defined levels of oxygen to which the organisms are exposed, using an online and near real-time acetylene reduction assay combined with sensitive laser-based photoacoustic ethylene detection. The cultures were grown under an alternating 12-12 h light-dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 mumol m(-2) s(-1) provided at the same light-dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5-7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29 degrees C. At 31 degrees C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41 degrees C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity

  8. How posttranslational modification of nitrogenase is circumvented in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively.

    PubMed

    Heiniger, Erin K; Oda, Yasuhiro; Samanta, Sudip K; Harwood, Caroline S

    2012-02-01

    Nitrogenase catalyzes the conversion of dinitrogen gas (N(2)) and protons to ammonia and hydrogen gas (H(2)). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris have been described that express nitrogenase genes constitutively and produce H(2) when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of nitrogenase. Microarray, mutation analysis, and gene expression studies showed that posttranslational regulation of nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated P(II) protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 nitrogenase modification enzyme. In addition, the NifA* strain had elevated nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H(2) production by an nifA* mutant grown with ammonium. Inactivation of the nitrogenase posttranslational modification system by mutation of draT2 resulted in increased H(2) production by ammonium-grown NifA* cells.

  9. Effect of Molybdenum Starvation and Tungsten on the Synthesis of Nitrogenase Components in Klebsiella pneumoniae

    PubMed Central

    Brill, Winston J.; Steiner, Ann L.; Shah, Vinod K.

    1974-01-01

    Klebsiella pneumoniae M5a1 grows well in the presence or absence of molybdenum in media containing excess NH4+. However, growth on N2 is completely dependent on the presence of molybdenum in the medium. Tungstate competes with the molybdate requirement during growth on N2. In molybdenum-depleted medium, neither protein component of nitrogenase is active and neither component can be detected antigenically. These data provide evidence that molybdenum is an inducer of nitrogenase synthesis. PMID:4598014

  10. Characterization of Diazotrophs Containing Mo-Independent Nitrogenases, Isolated from Diverse Natural Environments▿

    PubMed Central

    Betancourt, Doris A.; Loveless, Telisa M.; Brown, James W.; Bishop, Paul E.

    2008-01-01

    Molybdenum-independent nitrogenases were first described in the nitrogen-fixing bacterium Azotobacter vinelandii and have since been described in other diazotrophic bacteria. Previously, we reported the isolation of seven diazotrophs with Mo-independent nitrogenases from aquatic environments. In the present study, we extend these results to include diazotrophs isolated from wood chip mulch, soil, “paraffin dirt,” and sediments from mangrove swamps. Mo-deficient, N-free media under both aerobic and anaerobic conditions were used for the isolations. A total of 26 isolates were genetically and physiologically characterized. Their phylogenetic placement was determined using 16S rRNA gene sequence analysis. Most of the isolates are members of the gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria. Two other isolates, AN1 and LPF4, are closely related to Enterobacter spp. and Paenibacillus spp., respectively. PCR and/or Southern hybridization were used to detect the presence of nitrogenase genes in the isolates. PCR amplification of vnfG and anfG was used to detect the genetic potential for the expression of the vanadium-containing nitrogenase and the iron-only nitrogenase in the isolates. This study demonstrates that diazotrophs with Mo-independent nitrogenases can be readily isolated from diverse natural environments. PMID:18378646

  11. Seasonal changes in nodular nitrogenase activity of Alnus glutinosa and Elaeagnus angustifolia.

    PubMed

    Zitzer, S F; Dawson, J O

    1989-06-01

    Root nodule development, and seasonal patterns of nodular nitrogenase and hydrogenase activities were determined for 5- to 8-year old black alder (Alnus glutinosa (L.) Gaertn.) and Russian olive (Elaeagnus angustifolia L.) interplanted with black walnut (Juglans nigra L.) on bottomland and upland sites in central Illinois, USA. Black alder produced nodules at both sites, but Russian olive did so only at the bottomland site. Nodular nitrogenase activity was detectable in both species over a 220-day period. Maximum, midday rates of nitrogenase activity (acetylene reduction) of 15 to 20 micromoles C(2)H(4) per g dry nodule per hour were maintained by black alder for approximately 150 days at both the upland and bottomland sites. Near maximum rates of nodular nitrogenase activity were maintained for a similar period by Russian olive at the lowland site, although specific nitrogenase activity was approximately 25% lower than in black alder owing to a larger proportion of necrotic nodular tissue in Russian olive. In both species, nitrogenase activity increased exponentially with temperature between 10 degrees C and 20 to 25 degrees C. No net hydrogen evolution by nodules of either species was detected at any time during the assay period, indicating efficient hydrogenase systems were operating under the conditions of the field assay. Height of black walnut interplanted with nodulated black alder and Russian olive was greater than that of black walnut grown in pure stands.

  12. A survey of synthetic nicotinamide cofactors in enzymatic processes.

    PubMed

    Paul, Caroline E; Hollmann, Frank

    2016-06-01

    Synthetic nicotinamide cofactors are analogues of the natural cofactors used by oxidoreductases as redox intermediates. Their ability to be fine-tuned makes these biomimetics an attractive alternative to the natural cofactors in terms of stability, reactivity, and cost. The following mini-review focuses on the current state of the art of those biomimetics in enzymatic processes.

  13. Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration.

    PubMed

    Maiuri, Margherita; Delfino, Ines; Cerullo, Giulio; Manzoni, Cristian; Pelmenschikov, Vladimir; Guo, Yisong; Wang, Hongxin; Gee, Leland B; Dapper, Christie H; Newton, William E; Cramer, Stephen P

    2015-12-01

    We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226cm(-1) band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Recent trends and novel concepts in cofactor-dependent biotransformations.

    PubMed

    Kara, Selin; Schrittwieser, Joerg H; Hollmann, Frank; Ansorge-Schumacher, Marion B

    2014-02-01

    Cofactor-dependent enzymes catalyze a broad range of synthetically useful transformations. However, the cofactor requirement also poses economic and practical challenges for the application of these biocatalysts. For three decades, considerable research effort has been devoted to the development of reliable in situ regeneration methods for the most commonly employed cofactors, particularly NADH and NADPH. Today, researchers can choose from a plethora of options, and oxidoreductases are routinely employed even on industrial scale. Nevertheless, more efficient cofactor regeneration methods are still being developed, with the aim of achieving better atom economy, simpler reaction setups, and higher productivities. Besides, cofactor dependence has been recognized as an opportunity to confer novel reactivity upon enzymes by engineering their cofactors, and to couple (redox) biotransformations in multi-enzyme cascade systems. These novel concepts will help to further establish cofactor-dependent biotransformations as an attractive option for the synthesis of biologically active compounds, chiral building blocks, and bio-based platform molecules.

  15. Elimination of Rubisco alters the regulation of nitrogenase activity and increases hydrogen production in Rhodospirillum rubrum

    PubMed Central

    Wang, Di; Zhang, Yaoping; Welch, Emily; Li, Jilun; Roberts, Gary P.

    2010-01-01

    Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H2). The nitrogenase system is the primary means of H2 production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H2 production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO2 fixation pathway in R. rubrum induced nitrogenase activity even in the presence of ammonium, presumably to remove excess reductant in the cell. We report here the re-characterization of cbbM mutants in R. rubrum to study the effect of Rubisco on H2 production. Our newly constructed cbbM mutants grew poorly in malate medium under anaerobic conditions. However, the introduction of constitutively active NifA (NifA*), the transcriptional activator of the nitrogen fixation (nif) genes, allows cbbM mutants to dissipate the excess reductant through the nitrogenase system and improves their growth. Interestingly, we found that the deletion of cbbM alters the posttranslational regulation of nitrogenase activity, resulting in partially active nitrogenase in the presence of ammonium. The combination of mutations in nifA, draT and cbbM greatly increased H2 production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H2 over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H2. PMID:20652089

  16. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.

    PubMed

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-03-21

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

  17. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

    PubMed Central

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-01-01

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin–NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR–ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops. PMID:28193863

  18. Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

    PubMed Central

    Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

    2000-01-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  19. NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

    PubMed

    Hernandez, Jose A; Igarashi, Robert Y; Soboh, Basem; Curatti, Leonardo; Dean, Dennis R; Ludden, Paul W; Rubio, Luis M

    2007-01-01

    The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.

  20. Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6.

    PubMed

    Noar, Jesse; Loveless, Telisa; Navarro-Herrero, José Luis; Olson, Jonathan W; Bruno-Bárcena, José M

    2015-07-01

    The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. [Mechanisms for protecting nitrogenase from inactivation by oxygen].

    PubMed

    Soto-Urzúa, L; Baca, B E

    2001-01-01

    The biological fixation of dinitrogen is the most important way to access of N to organisms, this process requires a fairly high proportion of the ATP; which is generated in the course of respiratory electron transport reactions with O2 as electron acceptor. The Nitrogenase enzyme complex (the nitrogen. fixing enzyme) is sensitive to O2, that irreversible inactivates the enzyme. Diazotrophs must employ mechanisms which, on the other hand, permit the supply of O2 required for energy regeneration and protect Nase from the deleterious effect of O2. They have developed several strategies for limiting O2 access to Nase: 1).--It could avoid O2 and live in environments which are permanently anaerobic, 2).--Alternatively, it could generate a physical barrier around its Nase and in this way prevent O2 from diffusing to the enzyme, 3).--The microorganism could, by its metabolism, reduce the concentration of O2 within the vicinity of Nasa, 4).--They could modify its Nasa in such manner as to render it resistant to inactivation by O2 (conformational protection). 5).--Finally, the microorganism could simply balance Nasa inactivation with the synthesis of new enzyme. In this article we examine the antipathy between Nasa and O2, particularly with strict aerobic and photosynthetic microorganisms.

  2. Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

    PubMed Central

    Noar, Jesse; Loveless, Telisa; Navarro-Herrero, José Luis; Olson, Jonathan W.

    2015-01-01

    The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound. PMID:25911479

  3. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    PubMed Central

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  4. PBX proteins: much more than Hox cofactors.

    PubMed

    Laurent, Audrey; Bihan, Réjane; Omilli, Francis; Deschamps, Stéphane; Pellerin, Isabelle

    2008-01-01

    Pre-B cell leukaemia transcription factors (PBXs) were originally identified as Hox cofactors, acting within transcriptional regulation complexes to regulate genetic programs during development. Increasing amount of evidence revealed that PBX function is not restricted to a partnership with Hox or homeodomain proteins. Indeed, PBXs are expressed throughout murine embryonic development and are involved in several developmental pathways including Hox-independent mechanisms. This review summarizes what is known about PBX partnerships and proposes to position PBXs as central developmental factors whose role consists of integrating transduction signals, in order to regulate gene expression programs during development.

  5. First-principles calculations of Fischer-Tropsch processes catalyzed by nitrogenase enzymes

    NASA Astrophysics Data System (ADS)

    Varley, Joel; Grabow, Lars; Nørskov, Jens

    2012-02-01

    The nitrogenase enzyme system of the bacteria Azotobacter vinelandii, which is used in nature to catalyze ammonia synthesis, has been found recently to catalyze the efficient conversion of carbon monoxide (CO) into hydrocarbons under ambient temperature and pressure [1]. These findings indicate that nitrogenase enzymes could inspire more efficient catalysts for electrochemical CO and CO2 reduction to liquid fuels. The nitrogenase variants, in which vanadium substitutes the molybdenum in the active site of the enzyme, show distinct features in their reaction pathways to hydrocarbon production. To compare and contrast the catalytic properties of these nitrogenase enzymes, we perform first-principles calculations to map out the reaction pathways for both nitrogen fixation and for the reduction of CO to higher-order hydrocarbons. We discuss the trends and differences between the two enzymes and detail the relevant chemical species and rate-limiting steps involved in the reactions. By utilizing this information, we predict the electrochemical conditions necessary for the catalytic reduction of CO into fuels by the nitrogenase active sites, analogous to a Fischer-Tropsch process requiring less extreme conditions. [4pt] [1] Y. Hu, C.C. Lee, M.W. Ribbe, Science 333, 753 (2011)

  6. Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

    PubMed

    Sippel, Daniel; Schlesier, Julia; Rohde, Michael; Trncik, Christian; Decamps, Laure; Djurdjevic, Ivana; Spatzal, Thomas; Andrade, Susana L A; Einsle, Oliver

    2017-01-01

    The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

  7. N sub 2 O reduction and HD formation by nitrogenases from a nifV mutant of Klebsiella pneumoniae

    SciTech Connect

    Liang, J.; Burris, R.H. )

    1989-06-01

    Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The K{sub m}s of the mutant enzyme for N{sub 2} and N{sub 2}O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The K{sub m} for N{sub 2} was higher and the K{sub m} for N{sub 2}O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N{sub 2} fixation, in N{sub 2}O reduction, and in HD formation, as indicated by the low V{sub max} of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N{sub 2}, N{sub 2}O, and D{sub 2} interact with the same form of dinitrogenase. H{sub 2} evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H{sub 2} is released suggests that the mutant enzyme has more sites responsible for H{sub 2} evolution than the wild-type enzyme, whose H{sub 2} evolution is not inhibited by CO.

  8. Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome.

    PubMed

    Ivleva, Natalia B; Groat, Jeanna; Staub, Jeffrey M; Stephens, Michael

    2016-01-01

    Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers' expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids.

  9. Expression of Active Subunit of Nitrogenase via Integration into Plant Organelle Genome

    PubMed Central

    Groat, Jeanna; Staub, Jeffrey M.; Stephens, Michael

    2016-01-01

    Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers’ expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids. PMID:27529475

  10. Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase.

    PubMed

    Yang, Zhi-Yong; Moure, Vivian R; Dean, Dennis R; Seefeldt, Lance C

    2012-11-27

    A doubly substituted form of the nitrogenase MoFe protein (α-70(Val)(→Ala), α-195(His→Gln)) has the capacity to catalyze the reduction of carbon dioxide (CO(2)) to yield methane (CH(4)). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH(4) within 20 min. The catalytic rate depends on the partial pressure of CO(2) (or concentration of HCO(3)(-)) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H(2)C = CH-CH(3)) through the reductive coupling of CO(2) and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO(2) sequestration and formation of olefins.

  11. Reduction of N2 by supported tungsten clusters gives a model of the process by nitrogenase

    PubMed Central

    Murakami, Junichi; Yamaguchi, Wataru

    2012-01-01

    Metalloenzymes catalyze difficult chemical reactions under mild conditions. Mimicking their functions is a challenging task and it has been investigated using homogeneous systems containing metal complexes. The nitrogenase that converts N2 to NH3 under mild conditions is one of such enzymes. Efforts to realize the biological function have continued for more than four decades, which has resulted in several reports of reduction of N2, ligated to metal complexes in solutions, to NH3 by protonation under mild conditions. Here, we show that seemingly distinct supported small tungsten clusters in a dry environment reduce N2 under mild conditions like the nitrogenase. N2 is reduced to NH3 via N2H4 by addition of neutral H atoms, which agrees with the mechanism recently proposed for the N2 reduction on the active site of nitrogenase. The process on the supported clusters gives a model of the biological N2 reduction. PMID:22586517

  12. The in vivo hydrocarbon formation by vanadium nitrogenase follows a secondary metabolic pathway

    PubMed Central

    Rebelein, Johannes G.; Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W.

    2016-01-01

    The vanadium (V)-nitrogenase of Azotobacter vinelandii catalyses the in vitro conversion of carbon monoxide (CO) to hydrocarbons. Here we show that an A. vinelandii strain expressing the V-nitrogenase is capable of in vivo reduction of CO to ethylene (C2H4), ethane (C2H6) and propane (C3H8). Moreover, we demonstrate that CO is not used as a carbon source for cell growth, being instead reduced to hydrocarbons in a secondary metabolic pathway. These findings suggest a possible role of the ancient nitrogenase as an evolutionary link between the carbon and nitrogen cycles on Earth and establish a solid foundation for biotechnological adaptation of a whole-cell approach to recycling carbon wastes into hydrocarbon products. Thus, this study has several repercussions for evolution-, environment- and energy-related areas. PMID:27976719

  13. The in vivo hydrocarbon formation by vanadium nitrogenase follows a secondary metabolic pathway

    DOE PAGES

    Rebelein, Johannes G.; Lee, Chi Chung; Hu, Yilin; ...

    2016-12-15

    The vanadium (V)-nitrogenase of Azotobacter vinelandii catalyses the in vitro conversion of carbon monoxide (CO) to hydrocarbons. Here we show that an A. vinelandii strain expressing the V-nitrogenase is capable of in vivo reduction of CO to ethylene (C2H4), ethane (C2H6) and propane (C3H8). Moreover, we demonstrate that CO is not used as a carbon source for cell growth, being instead reduced to hydrocarbons in a secondary metabolic pathway. These findings suggest a possible role of the ancient nitrogenase as an evolutionary link between the carbon and nitrogen cycles on Earth and establish a solid foundation for biotechnological adaptation ofmore » a whole-cell approach to recycling carbon wastes into hydrocarbon products. Furthermore, this study has several repercussions for evolution-, environment- and energy-related areas.« less

  14. Exploring the intrinsic limits of nitrogenase transfer from bacteria to eukaryotes.

    PubMed

    Soto, Gabriela; Fox, Ana Romina; Ayub, Nicolás Daniel

    2013-08-01

    Biological nitrogen fixation is widespread among the Eubacteria and Archae domains but completely absent in eukaryotes. The lack of lateral transfer of nitrogen-fixation genes from prokaryotes to eukaryotes has been partially attributed to the physiological requirements necessary for the function of the nitrogenase complex. However, symbiotic bacterial nitrogenase activity is protected by the nodule, a plant structure whose organogenesis can be trigged in the absence of bacteria. To explore the intrinsic potentiality of this plant organ, we generated rhizobium-independent nodules in alfalfa by overexpressing the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the rhizobium-independent nodules can provide an optimal microenvironment for nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints on transfer of nitrogen-fixation genes between bacteria and eukaryotes.

  15. Requirement of homocitrate for the transfer of a 49V-labeled precursor of the iron-vanadium cofactor from VnfX to nif-apodinitrogenase.

    PubMed

    Ruttimann-Johnson, C; Rangaraj, P; Shah, V K; Ludden, P W

    2001-02-09

    A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium (Na(3)VO(4) or VCl(3)) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (alpha(2)beta(2)gamma(2)) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co-negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.

  16. Enhanced Nitrogenase Activity in Strains of Rhodobacter capsulatus That Overexpress the rnf Genes

    PubMed Central

    Jeong, Ho-Sang; Jouanneau, Yves

    2000-01-01

    In the photosynthetic bacterium Rhodobacter capsulatus, a putative membrane-bound complex encoded by the rnfABCDGEH operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole rnf operon was cloned under the control of the nifH promoter in plasmid pNR117 and expressed in several rnf mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in R. capsulatus. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex. PMID:10671439

  17. Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

    PubMed

    Haaker, H; Szafran, M; Wassink, H; Klerk, H; Appels, M

    1996-08-01

    The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

  18. Survey of the distribution of different types of nitrogenases and hydrogenases in heterocyst-forming cyanobacteria.

    PubMed

    Masukawa, Hajime; Zhang, Xiaohui; Yamazaki, Emi; Iwata, Syunsuke; Nakamura, Kensuke; Mochimaru, Mari; Inoue, Kazuhito; Sakurai, Hidehiro

    2009-01-01

    As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

  19. Pesticide side effect on the symbiotic efficiency and nitrogenase activity of Rhizobiaceae bacteria family.

    PubMed

    Niewiadomska, Alicja; Klama, Justyna

    2005-01-01

    The laboratory experiments tested the influence of selected pesticides on the symbiotic efficiency and nitrogenase activity of Rhizobium leguminosarumin bv. trifolii KGL, Sinorhizobiuni melilotii Bp and Badyrhizobium sp. Ornithopus B bacteria entering into symbiosis with clover, lucerne and serradella, respectively. The results obtained indicate that the pesticides used in the experiments (Funaben T seed dressing and Pivot 100SL herbicide) caused reduced nitrogenase activity in active strains tested. In addition, a toxic effect of the applied pesticides on the nodulation and root growth of the tested plants was observed.

  20. Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

    PubMed Central

    Ernst, A; Black, T; Cai, Y; Panoff, J M; Tiwari, D N; Wolk, C P

    1992-01-01

    Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane. Images PMID:1328150

  1. Importance of cis determinants and nitrogenase activity in regulated stability of the Klebsiella pneumoniae nitrogenase structural gene mRNA.

    PubMed

    Simon, H M; Gosink, M M; Roberts, G P

    1999-06-01

    The Klebsiella pneumoniae nitrogen fixation (nif) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30 degrees C). Addition of O2 or fixed nitrogen or temperature increases to 37 degrees C or more result in the dramatic destabilization of the nif mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of nif transcriptional regulation, was used to define cis determinants required for the regulated stability of the 5.2-kb nifHDKTY mRNA and to test the model suggested by earlier work that NifA is required in trans to stabilize nif mRNA under nif-derepressing conditions. O2 regulation of nifHDKTY mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of nifH, indicating that a site(s) required for the O2-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing nif mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of nifY mutants and overexpression suggests a possible involvement of the protein in this sensing process.

  2. Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-dependent and Cofactor-independent Examples

    PubMed Central

    Jordan, Frank; Patel, Hetalben

    2013-01-01

    This review is focused on three types of enzymes decarboxylating very different substrates: (1) Thiamin diphosphate (ThDP)-dependent enzymes reacting with 2-oxo acids; (2) Pyridoxal phosphate (PLP)-dependent enzymes reacting with α-amino acids; and (3) An enzyme with no known co-factors, orotidine 5'-monophosphate decarboxylase (OMPDC). While the first two classes have been much studied for many years, during the past decade studies of both classes have revealed novel mechanistic insight challenging accepted understanding. The enzyme OMPDC has posed a challenge to the enzymologist attempting to explain a 1017-fold rate acceleration in the absence of cofactors or even metal ions. A comparison of the available evidence on the three types of decarboxylases underlines some common features and more differences. The field of decarboxylases remains an interesting and challenging one for the mechanistic enzymologist notwithstanding the large amount of information already available. PMID:23914308

  3. Spectroscopic studies of molybdenum complexes as models for nitrogenase

    SciTech Connect

    Walker, T.P.

    1981-05-01

    Because biological nitrogen fixation requires Mo, there is an interest in inorganic Mo complexes which mimic the reactions of nitrogen-fixing enzymes. Two such complexes are the dimer Mo/sub 2/O/sub 4/ (cysteine)/sub 2//sup 2 -/ and trans-Mo(N/sub 2/)/sub 2/(dppe)/sub 2/ (dppe = 1,2-bis(diphenylphosphino)ethane). The H/sup 1/ and C/sup 13/ NMR of solutions of Mo/sub 2/O/sub 4/(cys)/sub 2//sup 2 -/ are described. It is shown that in aqueous solution the cysteine ligands assume at least three distinct configurations. A step-wise dissociation of the cysteine ligand is proposed to explain the data. The Extended X-ray Absorption Fine Structure (EXAFS) of trans-Mo(N/sub 2/)/sub 2/(dppe)/sub 2/ is described and compared to the EXAFS of MoH/sub 4/(dppe)/sub 2/. The spectra are fitted to amplitude and phase parameters developed at Bell Laboratories. On the basis of this analysis, one can determine (1) that the dinitrogen complex contains nitrogen and the hydride complex does not and (2) the correct Mo-N distance. This is significant because the Mo inn both complexes is coordinated by four P atoms which dominate the EXAFS. A similar sort of interference is present in nitrogenase due to S coordination of the Mo in the enzyme. This model experiment indicates that, given adequate signal to noise ratios, the presence or absence of dinitrogen coordination to Mo in the enzyme may be determined by EXAFS using existing data analysis techniques. A new reaction between Mo/sub 2/O/sub 4/(cys)/sub 2//sup 2 -/ and acetylene is described to the extent it is presently understood. A strong EPR signal is observed, suggesting the production of stable Mo(V) monomers. EXAFS studies support this suggestion. The Mo K-edge is described. The edge data suggests Mo(VI) is also produced in the reaction. Ultraviolet spectra suggest that cysteine is released in the course of the reaction.

  4. Nitrogenase activity in Trifolium subterraneum L. in relation to the uptake of nitrate ions. [Rhizobium trifolii

    SciTech Connect

    Silsbury, J.H.

    1987-07-01

    An experiment was conducted to test the hypothesis that, when nitrogenase and nitrate reductase both contribute to the nitrogen nutrition of a nodulated legume, nitrogenase activity is inversely proportional to the rate of accumulation of organic nitrogen derived from the reduction of nitrate. Trifolium subterraneum L. plants, inoculated with Rhizobium trifolii and sown as small swards, were allowed to establish a closed canopy and steady rates of growth, dinitrogen fixation, and nitrogen accumulation. Swards were then supplied with nutrient solutions of 0, 0.5, 1.0, or 2.5 mM NO/sub 3//sup -/ with a 29.69% enrichment of /sup 15/N and allowed to grow for a further 33 days. Harvests were made to measure dry weight, nitrogen accumulation, /sup 15/N accumulation, NO/sub 3//sup -/ content and nitrogenase activity by acetylene reduction assay. Since the /sup 15/N of the plant organic matter could have been derived only from the NO/sub 3//sup -/ of the nutrient solution, its rate of accumulation provided a measure of the rate of NO/sub 3//sup -/ reduction. It was found that as this rate increased in response to external NO/sub 3//sup -/ concentration the rate of nitrogenase activity decreased proportionately. It is concluded that the reduction of nitrate and the reduction of dinitrogen act in a complementary manner to supply a plant with organic nitrogen for growth.

  5. Effect of Oxygen and Malate on NO3− Inhibition of Nitrogenase in Soybean Nodules

    PubMed Central

    Heckmann, Marie-Odile; Drevon, Jean-Jacques; Saglio, Pierre; Salsac, Louis

    1989-01-01

    Soybean (Glycine max cv Hodgson) nitrogenase activity (C2H2 reduction) in the presence or absence of nitrate was studied at various external O2 tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O2 tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO2, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO2 increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O2. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis. PMID:16666740

  6. Influence of ammonium chloride on the nitrogenase activity of nodulated pea plants (Pisum sativum).

    PubMed Central

    Houwaard, F

    1978-01-01

    A study was made on the short-term effect of ammonium ions on the nitrogenase activity of pea root nodules. Nodulated pea plants (Pisum sativum), having reached maximum acetylene-reducing activity, were supplied with NH4Cl (20 mM). Nitrogenase activity of intact plants, detached nodules, and isolated bacteroids was measured at differed time intervals. A significant drop (20 to 40%) in the acetylene-reducing activity of treated intact plants and their detached nodules was observed after 1 day. No drop in the nitrogenase activity of bacteroids (assayed aerobically, or anaerobically after treatment with ethylenediaminetetraacetic acid-toluene) occurred for 2 to 4 days after the addition of NH4+ to the plants, depending on cultural conditions. From these results it is concluded that the adverse effect of NH4+ on acetylene reduction by intact plants and detached nodules during the first 2 days is not due to a decrease in the amount of nitrogenase in the bacteroids. It is suggested that the effect has to be attributed to a reduced supply to the bacteroids of energy-delivery photosynthates. PMID:677873

  7. Multiple recombination events maintain sequence identity among members of the nitrogenase multigene family in Rhizobium etli.

    PubMed Central

    Rodríguez, C; Romero, D

    1998-01-01

    A distinctive characteristic of the Rhizobium genome is the frequent finding of reiterated sequences, which often constitute multigene families. Interestingly, these families usually maintain a high degree of nucleotide sequence identity. It is commonly assumed that apparent gene conversion between reiterated elements might lead to concerted variation among members of a multigene family. However, the operation of this mechanism has not yet been demonstrated in the Rhizobiaceae. In this work, we employed different genetic constructions to address the role of apparent gene conversion as a homogenizing mechanism between members of the plasmid-located nitrogenase multigene family in Rhizobium etli. Our results show that a 28-bp insertion into one of the nitrogenase reiterations can be corrected by multiple recombination events, including apparent gene conversion. The correction process was dependent on the presence of both a wild-type recA gene and wild-type copies of the nitrogenase reiterations. Frequencies of apparent gene conversion to the wild-type nitrogenase reiterations were the same when the insertion to be corrected was located either in cis or in trans, indicating that this event frequently occurs through intermolecular interactions. Interestingly, a high frequency of multiple crossovers was observed, suggesting that these large plasmid molecules are engaging repeatedly in recombination events, in a situation akin to phage recombination or recombination among small, high-copy number plasmids. PMID:9611191

  8. Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening

    PubMed Central

    Barahona, Emma; Jiménez-Vicente, Emilio; Rubio, Luis M.

    2016-01-01

    When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases. PMID:27910898

  9. Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

    PubMed

    Barahona, Emma; Jiménez-Vicente, Emilio; Rubio, Luis M

    2016-12-02

    When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

  10. Temporal Variability in Nitrogenase Gene Expression in Natural Populations of the Marine Cyanobacterium Trichodesmium thiebautii

    PubMed Central

    Wyman, M.; Zehr, J. P.; Capone, D. G.

    1996-01-01

    We report a distinct diel periodicity in the abundance of nifH (dinitrogenase reductase) mRNA in natural populations of the nonheterocystous marine cyanobacterium Trichodesmium thiebautii. Our observations show that in addition to translational and posttranslational controls, Trichodesmium nitrogenase expression is also regulated at the transcriptional and/or posttranscriptional level. PMID:16535258

  11. Hydrothermal Synthesis and Structures of Two Tetramethylammonium Iron Molybdates (TMA) 2FeMo 6O 20and [TMA] 2[Fe(H 2O) 6]Mo 8O 26

    NASA Astrophysics Data System (ADS)

    Do, J.; Wang, X.; Jacobson, A. J.

    1999-02-01

    Two new compounds (TMA) 2FeMo 6O 20and [TMA] 2[Fe(H 2O) 6]Mo 8O 26have been synthesized by hydrothermal reactions. (TMA) 2FeMo 6O 20crystallizes in the monoclinic space group C2/ m; a=21.204(1) Å b=7.6393(5) Å, c=8.4191(6) Å, β=104.602(1)°, V=1319.7(2) Å 3, Z=2, ( R=2.23%, I>2 σ( I)) [TMA] 2[Fe(H 2O) 6]Mo 8O 26crystallizes in the space group P2 1/ n; a=10.3945(5) Å, b=16.4103(8) Å, c=10.8935(5) Å, β=98.842(1)°, V=1836.1(2) Å 3, Z=2 ( R=2.07% I>2 σ( I)). The structures of both compounds were determined by single crystal X-ray methods. The crystal structure of (TMA) 2FeMo 6O 20consists of 2∞[FeMo 6O 20] 2-layers separated by layers of tetramethylammonium cations. The [FeMo 6O 20] 2-layers are built up by the interconnection of corner- and edge-sharing MoO 6octahedral chains through FeO 6octahedra. The arrangement of MoO 6octahedra in the chains is identical to that found in the red potassium molybdenum bronze structure. In the (TMA) 2FeMo 6O 20structure, the chains are connected into layers by bridging FeO 6octahedra, in contrast to the bronze structure, where the chains are directly connected by sharing oxygen atoms. The structure of [TMA] 2[Fe(H 2O) 6]Mo 8O 26is made up by packing of octahedral [Fe(H 2O) 2+6] cations and β-[Mo 8O 4-26] cluster anions. These building units are interconnected through hydrogen bonds. Tetramethylammonium cations provide charge balance.

  12. Cofactor dependent conformational switching of GTPases.

    PubMed

    Hauryliuk, Vasili; Hansson, Sebastian; Ehrenberg, Måns

    2008-08-01

    This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP gamma S.

  13. Cofactor Dependent Conformational Switching of GTPases

    PubMed Central

    Hauryliuk, Vasili; Hansson, Sebastian; Ehrenberg, Måns

    2008-01-01

    This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\gamma}\\end{equation*}\\end{document} S. PMID:18502805

  14. Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

    PubMed Central

    Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos

    2014-01-01

    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. PMID:24244007

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

    PubMed

    Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos; Rubio, Luis M

    2014-02-01

    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.

  16. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid

    SciTech Connect

    Brown, K. A.; Harris, D. F.; Wilker, M. B.; Rasmussen, A.; Khadka, N.; Hamby, H.; Keable, S.; Dukovic, G.; Peters, J. W.; Seefeldt, L. C.; King, P. W.

    2016-04-21

    The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3. The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

  17. Control of nitrogenase recovery from oxygen inactivation by ammonia in the cyanobacterium Anabaena sp. strain CA (ATCC 33047).

    PubMed Central

    Smith, R L; Van Baalen, C; Tabita, F R

    1990-01-01

    The control of nitrogenase recovery from inactivation by oxygen was studied in Anabaena sp. strain CA (ATCC 33047). Nitrogenase activity (acetylene reduction) in cultures grown in 1% CO2 in air was inhibited by exposure to 1% CO2-99% O2 and allowed to recover in the presence of high oxygen tensions. Cultures exposed to hyperbaric levels of oxygen in the presence of 10 mM NH4NO3 were incapable of regaining nitrogenase activity, whereas control cultures returned to 65 to 80% of their original activity within about 3 h after exposure to high oxygen tension. In contrast to the regulation of heterocyst differentiation and nitrogenase synthesis, recovery from oxygen inactivation in this organism was shown to be under the control of NH4+ rather than NO3-. PMID:2110151

  18. In vivo generation of flavoproteins with modified cofactors.

    PubMed

    Mathes, Tilo; Vogl, Christian; Stolz, Jürgen; Hegemann, Peter

    2009-02-06

    To understand flavoprotein mechanisms and reactivity, biochemical and biophysical methods are usually employed, and differences between wild-type and mutated proteins with altered primary structures are placed under specific consideration. Alternatively, the cofactor can be modified, and modified flavoproteins can be studied accordingly. Here we present an efficient and general method for modifying the cofactor of flavoproteins in vivo. The modified cofactor is incorporated into apoprotein during protein biosynthesis in a riboflavin-auxotrophic Escherichia coli strain, which expresses a bacterial riboflavin transporter to import flavins from the medium. This system was used to introduce roseoflavin into the riboflavin-binding protein dodecin and into microbial blue-light photoreceptors of the BLUF (blue-light sensors using FAD) and LOV (light oxygen voltage) families. The modified photoreceptors showed absorption and fluorescence different from those of proteins carrying their natural cofactor or chromophores in solution, but did not show any photochemical reaction as implied by former physiological studies.

  19. Effect of nano-zinc oxide on nitrogenase activity in legumes: an interplay of concentration and exposure time

    NASA Astrophysics Data System (ADS)

    Kumar, Praveen; Burman, Uday; Santra, P.

    2015-07-01

    Experiments were carried out to study the effect of zinc oxide nanoparticles (nano-ZnO) on nitrogenase activity in legumes. In the first experiment, nodulated roots of cluster bean, moth bean, green gram and cowpea were dipped in Hoagland solution containing 1.5 and 10 μg mL-1 of nano-ZnO for 24 h. Nitrogenase activity in cluster bean, green gram and cowpea roots increased after dipping in solution containing 1.5 μg mL-1 nano-ZnO, but decreased in roots dipped in solution containing 10 μg mL-1 nano-ZnO. However, in moth bean roots, nitrogenase activity decreased after dipping in solution containing either concentration of nano-ZnO. In the second experiment, nodulated roots of green gram were dipped in Hoagland solution containing 1, 4, 6, 8 and 10 μg mL-1 nano-ZnO for 6-30 h before estimating nitrogenase activity. Results showed that an interactive effect of nano-ZnO concentration and exposure time influenced nitrogenase activity. The possible reasons behind this effect have been discussed. A model [ A = 3.44 + 0.46 t - 0.01 t 2 - 0.002 tc 2 ( R 2 = 0.81)] involving linear and power components was developed to simulate the response of nitrogenase activity in green gram roots to the concentration and exposure time of nano-ZnO.

  20. Application of the photoacoustic method to the measurement of acetylene reduction by nitrogenase enzyme

    NASA Astrophysics Data System (ADS)

    Schramm, D. U.; Sthel, M. S.; Carneiro, L. O.; Franco, A. A.; Campos, A. C.; Vargas, H.

    2005-06-01

    Nitrogenase is an enzyme responsible for the reduction of the atmospheric N2 into NH4^+, which represents the key entry point of the molecular nitrogen into the biogeochemical cycle of nitrogen. This enzyme is present in the rhizobial bacteroids, which are symbionts in a Leguminosae plant (Acacia Holosericea), and also reduces acetylene into ethylene at the same rate as the nitrogen reduction. Therefore, a CO2 Laser Photoacoustic system was used for detecting and monitoring the ethylene emission by the nitrogenase activity, in the rhizobial symbionts in Acacia Holosericea, when they are confined in test tubes with acetylene at two different volumes (0.1 and 0.5 ml). Ethylene concentrations are also determined in the ppm range.

  1. Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides

    PubMed Central

    Farmer, Ryan M.

    2015-01-01

    In many organisms there is a balance between carbon and nitrogen metabolism. These observations extend to the nitrogen-fixing, nonsulfur purple bacteria, which have the classic family of P(II) regulators that coordinate signals of carbon and nitrogen status to regulate nitrogen metabolism. Curiously, these organisms also possess a reverse mechanism to regulate carbon metabolism based on cellular nitrogen status. In this work, studies in Rhodobacter sphaeroides firmly established that the activity of the enzyme that catalyses nitrogen fixation, nitrogenase, induces a signal that leads to repression of genes encoding enzymes of the Calvin–Benson–Bassham (CBB) CO2 fixation pathway. Additionally, genetic and metabolomic experiments revealed that NADH-activated phosphoribulokinase is an intermediate in the signalling pathway. Thus, nitrogenase activity appears to be linked to cbb gene repression through phosphoribulokinase. PMID:26306848

  2. Crystallographic studies of nitrogenase and hydrogenase. Progress report, June 1, 1992--April 1, 1994

    SciTech Connect

    Bolin, J.T.

    1994-05-01

    The long term goal of this project is to obtain detailed knowledge of the structure and function of nitrogenase and hydrogenase through the analysis of physical, chemical, and biological data with reference to three-dimensional, atomic resolution crystal structures of components of the enzyme and/or complexes of the components. The current objectives to determine the crystal structure of wild-type Av1, the nitrogenase MoFe protein from Azotobacter vinelandii; to refine this structure at high resolution; and to initiate studies of mutant MoFe proteins that express altered chemical and physical properties. Further we seek to determine the crystal structure of the bi-directional all-Fe hydrogenase from C. pasteurianum, Cp-hydrI, and to initiate studies of the uptake hydrogenase from the same organism, Cp-hydrII.

  3. Regulatory effect of hydrogen on nitrogenase activity of the blue-green alga (cyanobacterium) Nostoc muscorum.

    PubMed

    Scherer, S; Kerfin, W; Böger, P

    1980-03-01

    Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under an atmosphere of argon plus acetylene in the light led to a greater than fourfold increase of light-induced hydrogen evolution and to a 50% increase of acetylene reduction, as compared to cells that had not been preconditioned. The basic and the increased hydrogen evolution were both due to nitrogenase activity. Furthermore, after preincubation the hydrogen uptake, usually observed with unconditional cells, was abolished. Nostoc preincubated under acetylene evolved hydrogen in the light even in the presence of nitrogen for at least 2 h, with a 15-fold increase as compared to the unconditioned cells. These acetylene effects could be completely abolished by the presence of hydrogen during acetylene preincubation. These findings indicate that the hydrogen concentration in N. muscorum cells plays a role in regulation of nitrogenase activity.

  4. Nitrite and hydroxylamine as nitrogenase substrates: mechanistic implications for the pathway of N₂ reduction.

    PubMed

    Shaw, Sudipta; Lukoyanov, Dmitriy; Danyal, Karamatullah; Dean, Dennis R; Hoffman, Brian M; Seefeldt, Lance C

    2014-09-10

    Investigations of reduction of nitrite (NO2(-)) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2(-) + 6e(-) + 12ATP + 7H(+) → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2(-) turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2(-) reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2(-) reductions in light of their common reduction intermediates and of NO2(-) reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2(-) reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e(-)/H(+)] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M-[NO(+)]; transfer of the E2 hydride to the [NO(+)] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M-[NO] thermodynamic "sink". The N2 and NO2(-) reduction pathways converge upon reduction of NH2NH2 and NH2OH bound states to form state H with [-NH2] bound to M. Final reduction converts H to I, with NH3 bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH3 occurs upon delivery of five [e(-)/H(+)] to N2, but a total of seven [e(-)/H(+)] to FeMo-co when obligate H2 evolution is considered, and not earlier in the reduction process.

  5. Modulating the midpoint potential of the [4Fe-4S] cluster of the nitrogenase Fe protein.

    PubMed

    Jang, S B; Seefeldt, L C; Peters, J W

    2000-02-01

    Protein-bound [FeS] clusters function widely in biological electron-transfer reactions, where their midpoint potentials control both the kinetics and thermodynamics of these reactions. The polarity of the protein environment around [FeS] clusters appears to contribute largely to modulating their midpoint potentials, with local protein dipoles and water dipoles largely defining the polarity. The function of the [4Fe-4S] cluster containing Fe protein in nitrogenase catalysis is, at least in part, to serve as the nucleotide-dependent electron donor to the MoFe protein which contains the sites for substrate binding and reduction. The ability of the Fe protein to function in this manner is dependent on its ability to adopt the appropriate conformation for productive interaction with the MoFe protein and on its ability to change redox potentials to provide the driving force required for electron transfer. Phenylalanine at position 135 is located near the [4Fe-4S] cluster of nitrogenase Fe protein and has been suggested by amino acid substitution studies to participate in defining both the midpoint potential and the nucleotide-induced changes in the [4Fe-4S] cluster. In the present study, the crystal structure of the Azotobacter vinelandii nitrogenase Fe protein variant having phenylalanine at position 135 substituted by tryptophan has been determined by X-ray diffraction methods and refined to 2.4 A resolution. A comparison of available Fe protein structures not only provides a structural basis for the more positive midpoint potential observed in the tryptophan substituted variant but also suggests a possible general mechanism by which the midpoint potential could be controlled by nucleotide interactions and nitrogenase complex formation.

  6. Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

    PubMed

    Burén, Stefan; Young, Eric M; Sweeny, Elizabeth A; Lopez-Torrejón, Gema; Veldhuizen, Marcel; Voigt, Christopher A; Rubio, Luis M

    2017-06-16

    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.

  7. Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness.

    PubMed

    Selao, Tiago Toscano; Edgren, Tomas; Wang, He; Norén, Agneta; Nordlund, Stefan

    2011-06-01

    Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to P(II) protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

  8. Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

    PubMed Central

    2017-01-01

    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. PMID:28221768

  9. Efficiently Communicating Rich Heterogeneous Geospatial Data from the FeMO2008 Dive Cruise with FlashMap on EarthRef.org

    NASA Astrophysics Data System (ADS)

    Minnett, R. C.; Koppers, A. A.; Staudigel, D.; Staudigel, H.

    2008-12-01

    the web without losing scalability and control of the base maps. Our Flash-based application is fully compatible with KML (Keyhole Markup Language) 2.2, the most recent iteration of KML, allowing users with existing Google Earth KML files to effortlessly display their geospatial content embedded in a web page. As a test case for FlashMap, the annual Iron-Oxidizing Microbial Observatory (FeMO) dive cruise to the Loihi Seamount, in conjunction with data available from ongoing and published FeMO laboratory studies, showcases the flexibility of this single web-based application. With a KML 2.2 compatible web-service providing the content, any database can display results in FlashMap. The user can then hide and show multiple layers of content, potentially from several data sources, and rapidly digest a vast quantity of information to narrow the search results. This flexibility gives experienced users the ability to drill down to exactly the record they are looking for (SERC at Carleton College's educational application of FlashMap at http://serc.carleton.edu/sp/erese/activities/22223.html) and allows users familiar with Google Earth the ability to load and view geospatial data content within a browser from any computer with an internet connection.

  10. Position of the ATP-binding site of the Fe-protein relative to the iron-sulfur clusters 4Fe-4S and the iron-molybdenum-containing cofactor

    SciTech Connect

    Kondrat'eva, T.A.; Gvozdev, R.I.; Mitsova, I.Z.

    1986-06-10

    Nitrogenase was affinity labeled with epsilon-ATP at the ATP-binding sites and separated into protein components by ion exchange chromatography. In spectrofluorometric titration of the labeled Fe-protein with the native MoFe-protein from the wild strain of Azotobacter and the MoFe-protein not containing iron-sulfur clusters 4Fe-4S, a 4-6-fold quenching of the fluorescence of immobilized epsilon-ATP was observed. When the labeled Fe-protein was titrated with MoFe-protein from the Azotobacter mutant UW-45, on the contrary, there was a four-fold increase in the fluorescence of immobilized epsilon-ATP. Since the MoFe-protein of the Azotobacter mutant UW-45 differs from the MoFe-protein from the wild strain of Azotobacter only by the absence of an iron-molybdenum-containing cofactor (Fe-Mo-cofactor), it is suggested that the ATP-binding site of the Fe-protein is situated next to the FeMo-cofactor and at a distance from the iron-sulfur clusters 4Fe-4S when a complex is formed with the MoFe-protein. The formation of a complex is accompanied by a change in the conformation of the Fe-protein.

  11. Mechanism of nitrogenase switch-off by oxygen. [Klebsiella pneumoniae; Rhodopseudomonas sphaeroides f. sp. denitrificans; Rhodopseudomonas capsulate

    SciTech Connect

    Goldberg, I.; Nadler, V.; Hochman, A.

    1987-02-01

    Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strians AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O/sub 2/ concentration of 0.37 ..mu..M. Cyanide (90 ..mu..M), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase activity to 2.9 ..mu..M. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but is nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 ..mu..M O/sub 2/. Inhibition by CN/sup -/ of 40 to 50% of the O/sub 2/ uptake in the mutant shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase to 1.58 ..mu..M. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO/sub 2//sup -/. Lower concentrations of NO/sub 2//sup -/ were required to inhibit the activity in NO/sub 3//sup -/-grown cells, which have higher activities of nitrite reductase.

  12. Cofactor Engineering for Enhancing the Flux of Metabolic Pathways

    PubMed Central

    Akhtar, M. Kalim; Jones, Patrik R.

    2014-01-01

    The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering. PMID:25221776

  13. Emerging roles for tubulin folding cofactors at the centrosome

    PubMed Central

    Fanarraga, Mónica López; Carranza, Gerardo; Castaño, Raquel; Jiménez, Victoria; Villegas, Juan Carlos

    2010-01-01

    Despite its fundamental role in centrosome biology, procentriole formation, both in the canonical and in the de novo replication pathways, remains poorly understood, and the molecular components that are involved in human cells are not well established. We found that one of the tubulin cofactors, TBCD, is localized at centrosomes and the midbody, and is required for spindle organization, cell abscission, centriole formation and ciliogenesis. Our studies have established a molecular link between the centriole and the midbody, demonstrating that this cofactor is also necessary for microtubule retraction during cell abscission. TBCD is the first centriolar protein identified that plays a role in the assembly of both “centriolar rosettes” during early ciliogenesis, and at the procentriole budding site by S/G2, a discovery that directly implicates tubulin cofactors in the cell division, cell migration and cell signaling research fields. PMID:20798813

  14. The chemical mechanism of nitrogenase: calculated details of the intramolecular mechanism for hydrogenation of eta(2)-N(2) on FeMo-co to NH(3).

    PubMed

    Dance, Ian

    2008-11-21

    Using density functional calculations, a complete chemical mechanism has been developed for the reaction N(2) + 6e(-) + 6H(+)--> 2NH(3) catalyzed by the Fe(7)MoS(9)N(c)(homocitrate) cofactor (FeMo-co) of the enzyme nitrogenase. The mechanism is based on previous descriptions of the generation of H atoms on FeMo-co by proton relay through a protein path terminating in water molecule 679, and preserves the model (which explains much biochemical data) for vectorial migration of H atoms to two S atoms and two Fe atoms of FeMo-co. After calculation of the energy profiles for the many possible sequences of steps in which these H atoms are transferred to N(2) and its hydrogenated intermediates, a favourable pathway to 2NH(3) was developed. Transition states and activation potential energies for the 21 step mechanism are presented, together with results for some alternative branches. The mechanism develops logically from the eta(2)-coordination of N(2) at the endo position of one Fe atom of prehydrogenated FeMo-co, consistent with the previous kinetic-mechanistic scheme of Thorneley and Lowe, and passes through bound N(2)H(2) and N(2)H(4) intermediates. This mechanism is different from others in the literature because it uses a single replenishable path for serial supply of protons which become H atoms on FeMo-co, migrating to become S-H and Fe-H donors to N(2) and to the intermediates that follow. The new paradigm for the chemical catalysis is that hydrogenation of N(2) and intermediates is intramolecular and does not involve direct protonation from surrounding residues which appear to be unable to provide a replenishable supply of 6H(+). Many steps in this intramolecular hydrogenation are expected to be enhanced by H tunneling.

  15. Isotopic hybrids of nitrogenase. Mössbauer study of MoFe protein with selective 57Fe enrichment of the P-cluster.

    PubMed

    McLean, P A; Papaefthymiou, V; Orme-Johnson, W H; Münck, E

    1987-09-25

    Previous Mössbauer and EPR studies of the MoFe protein (approximately 30 Fe and 2 Mo) of nitrogenase have revealed the presence of two unique clusters, namely, the P-clusters (presumably of the Fe4S4 type) and the molybdenum- and iron-containing cofactors (or M-clusters). Mössbauer components D (approximately 10-12 Fe) and Fe2+ (approximately 4 Fe) represent subsites of the P-clusters while component S (approximately 2 Fe) appeared to belong to a separate, unidentified cluster. In order to refine the analyses of Mössbauer spectra, we have constructed an isotopic hybrid of the Klebsiella pneumoniae protein which contains 57Fe-enriched P-clusters and 56Fe-enriched M-clusters. The highly resolved 57Fe Mössbauer spectra of this hybrid show that component S behaves spectroscopically like the P-cluster sites D and Fe2+ in oxidized and reduced MoFe protein. This suggests that S is a subset of the P-clusters rather than a different cluster type. The present study shows, for the first time, that the Debye-Waller factors of different P-cluster subsites have a different temperature dependence. Thus, the Fe2+/D absorption ratio is 4.0:10.0 at 4.2 K and 4.0:11.6 at 173 K. We propose that the reduced MoFe protein contains two pairs of P-clusters: one pair containing one Fe2+ and three D-sites and the other one Fe2+, two D, and one S-site. We have argued previously that the oxidized P-clusters occur in pairs as well.

  16. Pterin chemistry and its relationship to the molybdenum cofactor

    PubMed Central

    Basu, Partha; Burgmayer, Sharon J.N.

    2011-01-01

    The molybdenum cofactor is composed of a molybdenum coordinated by one or two rather complicated ligands known as either molybdopterin or pyranopterin. Pterin is one of a large family of bicyclic N-heterocycles called pteridines. Such molecules are widely found in Nature, having various forms to perform a variety of biological functions. This article describes the basic nomenclature of pterin, their biological roles, structure, chemical synthesis and redox reactivity. In addition, the biosynthesis of pterins and current models of the molybdenum cofactor are discussed. PMID:21607119

  17. Generation of protein-derived redox cofactors by posttranslational modification.

    PubMed

    Davidson, Victor L

    2011-01-01

    Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.

  18. Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142

    PubMed Central

    Bernstein, Hans C.; Charania, Moiz A.; McClure, Ryan S.; Sadler, Natalie C.; Melnicki, Matthew R.; Hill, Eric A.; Markillie, Lye Meng; Nicora, Carrie D.; Wright, Aaron T.; Romine, Margaret F.; Beliaev, Alexander S.

    2015-01-01

    To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process. PMID:26525576

  19. New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes

    SciTech Connect

    Zehr, J.P.; Mellon, M.T.; Zani, S.

    1998-09-01

    Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the {alpha} and {gamma} subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.

  20. New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of Nitrogenase (nifH) genes.

    PubMed

    Zehr, J P; Mellon, M T; Zani, S

    1998-09-01

    Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the alpha and gamma subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.

  1. The in vivo hydrocarbon formation by vanadium nitrogenase follows a secondary metabolic pathway

    SciTech Connect

    Rebelein, Johannes G.; Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W.

    2016-12-15

    The vanadium (V)-nitrogenase of Azotobacter vinelandii catalyses the in vitro conversion of carbon monoxide (CO) to hydrocarbons. Here we show that an A. vinelandii strain expressing the V-nitrogenase is capable of in vivo reduction of CO to ethylene (C2H4), ethane (C2H6) and propane (C3H8). Moreover, we demonstrate that CO is not used as a carbon source for cell growth, being instead reduced to hydrocarbons in a secondary metabolic pathway. These findings suggest a possible role of the ancient nitrogenase as an evolutionary link between the carbon and nitrogen cycles on Earth and establish a solid foundation for biotechnological adaptation of a whole-cell approach to recycling carbon wastes into hydrocarbon products. Furthermore, this study has several repercussions for evolution-, environment- and energy-related areas.

  2. Effect of sulphur dioxide exposure on chlorophyll content and nitrogenase activity of Vicia faba L. plants

    SciTech Connect

    Agrawal, S.B.; Agrawal, M. )

    1991-11-01

    The annual average concentrations of SO{sub 2} around Obra thermal power plant and nonpolluted sites in India were reported as 0.06, and 0.007 ppm, respectively. However, daily average concentrations in areas close to the emission source may be as large as 0.34 ppm. Therefore, in the present investigation an attempt has been made to determine the potential effects of such episodic and exceptionally high intermittent concentrations of SO{sub 2} on total chlorophyll content and nitrogenase activity of Vicia faba (broad bean) plants.

  3. Citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumoniae

    SciTech Connect

    Liang, Jihong; Madden, M.; Shah, V.K.; Burris, R.H. )

    1990-09-18

    An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiella pneumoniae has been identified as citric acid. H{sub 2} evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV{sup {minus}} dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.

  4. Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism.

    PubMed Central

    Smith, B E; Thorneley, R N; Eady, R R; Mortenson, L E

    1976-01-01

    In combination with the Mo-Fe protein of nitrogenase from Klebsiella pneumoniae, the Fe protein of nitrogenase from Clostridium pasteurianum forms an active enzyme with novel properties different from those of either of the homologous nitrogenases. The steady-state rates of reduction of acetylene and H+ are 12% of those of the homologous system from C.pasteurianim. Acetylene reductase activity exhibited an approx. 10min lag at 30 degrees C before the rate of reduction became linear, consistent with a once-only activation step being necessary for acetylene reduction to occur. No such lag was observed for H2 evolution. The activity with N2 as a reducible substrate was very low, implying that acetylene reductase activity is not necessarily an accurate indication of nitrogen-fixing ability. This is of particular relevance to studies on mutant and agronomically important organisms. Stopped-flow spectrophotometric studies showed unimolecular electron transfer from the Fe protein to the Mo-Fe protein to occur at the same rate (k2 = 2.5 X 10(2)s-1) and with the same dependence on ATP concentration (apparent KD = 400 muM) as with the homologous Klebsiella nitrogenase. However, an ATP/2e ratio of 50 was obtained for H2 evolution, indicating that ATP hydrolysis had been uncoupled from electron transfer to substrate. These data indicate that ATP has at least two roles in the mechanism of nitrogenase action. The combination of the Mo-Fe protein of nitrogenase of C.pasteurianim and the Fe protein of K.pneumoniae were inactive in all the above reactions, except for a weak adenosine triphosphatase activity, 0.5% of that of the homologous K.pneumoniae system. Images Fig. 3. PMID:134700

  5. Dendrite arborization requires the dynein cofactor NudE.

    PubMed

    Arthur, Ashley L; Yang, Sihui Z; Abellaneda, Allison M; Wildonger, Jill

    2015-06-01

    The microtubule-based molecular motor dynein is essential for proper neuronal morphogenesis. Dynein activity is regulated by cofactors, and the role(s) of these cofactors in shaping neuronal structure are still being elucidated. Using Drosophila melanogaster, we reveal that the loss of the dynein cofactor NudE results in abnormal dendrite arborization. Our data show that NudE associates with Golgi outposts, which mediate dendrite branching, suggesting that NudE normally influences dendrite patterning by regulating Golgi outpost transport. Neurons lacking NudE also have increased microtubule dynamics, reflecting a change in microtubule stability that is likely to also contribute to abnormal dendrite growth and branching. These defects in dendritogenesis are rescued by elevating levels of Lis1, another dynein cofactor that interacts with NudE as part of a tripartite complex. Our data further show that the NudE C-terminus is dispensable for dendrite morphogenesis and is likely to modulate NudE activity. We propose that a key function of NudE is to enhance an interaction between Lis1 and dynein that is crucial for motor activity and dendrite architecture.

  6. Chemistry and bioactivity of an artificial adenosylpeptide B(12) cofactor.

    PubMed

    Zhou, Kai; Oetterli, René M; Brandl, Helmut; Lyatuu, Fredrick E; Buckel, Wolfgang; Zelder, Felix

    2012-09-24

    Artificial influence: We describe a semi-artificial adenosylpeptide B(12) that behaves as a cofactor in B(12)-dependent enzymatic reactions and demonstrate that the peptide backbone influences its chemical properties and modulates its bioactivity in vitro and in vivo. Inhibition of the growth of L. delbrueckii is demonstrated, thus providing a potentially powerful approach for the development of antibacterial and antiproliferative compounds.

  7. Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif- mutants.

    PubMed Central

    Imperial, J; Shah, V K; Ugalde, R A; Ludden, P W; Brill, W J

    1987-01-01

    Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants. PMID:3470286

  8. Cofactor Trapping, a New Method To Produce Flavin Mononucleotide ▿

    PubMed Central

    Krauss, Ulrich; Svensson, Vera; Wirtz, Astrid; Knieps-Grünhagen, Esther; Jaeger, Karl-Erich

    2011-01-01

    We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN. PMID:21131527

  9. Studies on free radicals, antioxidants, and co-factors

    PubMed Central

    Rahman, Khalid

    2007-01-01

    The interplay between free radicals, antioxidants, and co-factors is important in maintaining health, aging and age-related diseases. Free radicals induce oxidative stress, which is balanced by the body’s endogenous antioxidant systems with an input from co-factors, and by the ingestion of exogenous antioxidants. If the generation of free radicals exceeds the protective effects of antioxidants, and some co-factors, this can cause oxidative damage which accumulates during the life cycle, and has been implicated in aging, and age dependent diseases such as cardiovascular disease, cancer, neurodegenerative disorders, and other chronic conditions. The life expectancy of the world population is increasing, and it is estimated that by 2025, 29% of the world population will be aged ≥60 years, and this will lead to an increase in the number of older people acquiring age-related chronic diseases. This will place greater financial burden on health services and high social cost for individuals and society. In order to acheive healthy aging the older people should be encouraged to acquire healthy life styles which should include diets rich in antioxidants. The aim of this review is to highlight the main themes from studies on free radicals, antioxidants and co-factors, and to propose an evidence-based strategy for healthy aging. PMID:18044138

  10. Recognition of enzymes lacking bound cofactor by protein quality control

    PubMed Central

    Martínez-Limón, Adrián; Alriquet, Marion; Lang, Wei-Han; Calloni, Giulia; Wittig, Ilka; Vabulas, R. Martin

    2016-01-01

    Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor binding-related enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders. PMID:27733512

  11. Structural Basis for Cofactor-Independent Dioxygenation in Vancomycin Biosynthesis

    SciTech Connect

    Widboom,P.; Fielding, E.; Liu, Y.; Bruner, S.

    2007-01-01

    Enzyme-catalyzed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion1. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.

  12. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr

    NASA Astrophysics Data System (ADS)

    Stüeken, Eva E.; Buick, Roger; Guy, Bradley M.; Koehler, Matthew C.

    2015-04-01

    Nitrogen is an essential nutrient for all organisms that must have been available since the origin of life. Abiotic processes including hydrothermal reduction, photochemical reactions, or lightning discharge could have converted atmospheric N2 into assimilable NH4+, HCN, or NOx species, collectively termed fixed nitrogen. But these sources may have been small on the early Earth, severely limiting the size of the primordial biosphere. The evolution of the nitrogen-fixing enzyme nitrogenase, which reduces atmospheric N2 to organic NH4+, thus represented a major breakthrough in the radiation of life, but its timing is uncertain. Here we present nitrogen isotope ratios with a mean of 0.0 +/- 1.2‰ from marine and fluvial sedimentary rocks of prehnite-pumpellyite to greenschist metamorphic grade between 3.2 and 2.75 billion years ago. These data cannot readily be explained by abiotic processes and therefore suggest biological nitrogen fixation, most probably using molybdenum-based nitrogenase as opposed to other variants that impart significant negative fractionations. Our data place a minimum age constraint of 3.2 billion years on the origin of biological nitrogen fixation and suggest that molybdenum was bioavailable in the mid-Archaean ocean long before the Great Oxidation Event.

  13. Early evolution of photosynthesis: Clues from nitrogenase and chlorophyll iron proteins

    SciTech Connect

    Burke, D.H.; Hearst, J.E.; Sidow, A. )

    1993-08-01

    Chlorophyll (Chl) is often viewed as having preceded bacteriochlorophyll (BChl) as the primary photoreceptor pigment in early photosynthetic systems because synthesis of Chl requires one fewer enzymatic reduction than does synthesis of BChl. The authors have conducted statistical DNA sequence analyses of the two reductases involved in Chl and BChl synthesis, protochlorophyllide reductase and chlorin reductase. Both are three-subunit enzymes in which each subunit from one reductase shares significant amino acid identity with a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The [open quotes]chlorophyll iron protein[close quotes] subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, also share amino acid sequence identity with the nitrogenase iron protein, encoded by nifH. When nitrogenase iron proteins are used as outgroups, the chlorophyll iron protein tree is rooted on the chlorin reductase lineage. This rooting suggests that the last common ancestor of all extant photosynthetic eubacteria contained BChl, not Chl, in its reaction center, and implies that Chl-containing reaction centers were a late invention unique to the cyanobacteria/chloroplast lineage. 48 refs., 4 figs., 2 tabs.

  14. Influence of different factors on the nitrogenase activity of the engineered Escherichia coli 78-7.

    PubMed

    Zhang, Li-hong; Chen, San-feng

    2015-06-01

    The engineered Escherichia coli 78-7 is a derivative of E. coli JM109 carrying a nitrogen fixation (nif) gene cluster composed of nine genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) and its own σ(70)-dependent nif promoter from a gram-positive bacterium Paenibacillus sp. WLY78. The physiological and biochemical characteristics of the engineered E. coli 78-7 were analyzed by using Biolog GEN III MicroPlate, with E. coli JM109 and JM109/pHY300PLK (E. coli JM109 carrying empty vector) as controls. Analysis of 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity tests showed that the engineered E. coli 78-7, E. coli JM109 and JM109/pHY300PLK gave similar patterns of utilization of various substrates as carbon and energy sources. Furthermore, the effect of carbon source, nitrogen source, culture temperature on the nitrogenase activity of the engineered E. coli 78-7 was investigated. Our study demonstrates that the nif capacity of E. coli 78-7 was affected significantly by the different culture condition. The significant nitrogenase activity of E. coli 78-7 were obtained when cells were cultivated in the medium containing 4 g/l glucose (carbon source) and 2 mM glutamate (nitrogen source) and at 30 °C.

  15. Surface disturbance of cryptobiotic soil crusts: nitrogenase activity, chlorophyll content, and chlorophyll degradation

    USGS Publications Warehouse

    Belnap, Jayne; Harper, Kimball T.; Warren, Steven D.

    1994-01-01

    Cryptobiotic soil crusts are an important component of semiarid and arid ecosystems. An important role of these crusts is the contribution of fixed nitrogen to cold‐desert ecosystems. This study examines the residual effects of various intensities and combinations of different surface disturbances (raking, scalping, and tracked vehicles) on nitrogenase activity, chlorophyll content, and chlorophyll degradation in these soil crusts. Nine months after disturbance chlorophyll content of disturbed soils was not statistically different from undisturbed controls, except in the scalped treatments, indicating recovery of this characteristic is fairly quick unless surface material is removed. Differences in chlorophyll degradation among treatments were not statistically significant. However, nitrogenase activity in all treatments showed tremendous reductions, ranging from 77–97%, when compared to the control, indicating this characteristic is slow to recover. Consequently, assessment of crustal recovery from disturbance must include not only visual and biomass characteristics but other physiological measurements as well. Areas dominated by these crusts should be managed conservatively until the implications of crustal disturbance is better understood.

  16. Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea.

    PubMed

    Kim, Chungwoo; Kecskés, Mihály L; Deaker, Rosalind J; Gilchrist, Kate; New, Peter B; Kennedy, Ivan R; Kim, Seunghwan; Sa, Tongmin

    2005-11-01

    Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.

  17. Nodule-Specific Polypeptides from Effective Alfalfa Root Nodules and from Ineffective Nodules Lacking Nitrogenase 1

    PubMed Central

    Lang-Unnasch, Naomi; Ausubel, Frederick M.

    1985-01-01

    In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664146

  18. Involvement of GroEL in nif gene regulation and nitrogenase assembly.

    PubMed

    Govezensky, D; Greener, T; Segal, G; Zamir, A

    1991-10-01

    Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.

  19. Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants 1

    PubMed Central

    De-Polli, Helvecio; Boyer, Charles D.; Neyra, Carlos A.

    1982-01-01

    Corn (Zea mays L.) plants were assayed for nitrogenase activity (C2H2 reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O2 resulted in lower rates of C2H2 reduction. The time course of C2H2 reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn × teosinte) and indicated that a long preincubation at reduced O2 is not absolutely required for early detection of nitrogenase activity. Isolation of N2-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N2-fixing bacteria. PMID:16662729

  20. Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules

    SciTech Connect

    Monroe, J.D. ); LaRue, T.A. )

    1989-10-01

    The interaction between carbon substrates and O{sub 2} and their effects on nitrogenase activity (C{sub 2}H{sub 2}) were examined in detached nodules of pea (Pisum sativum L. cv Sparkle). The internal O{sub 2} concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O{sub 2} for 2 hours resulted in a 2- to 10-fold increase in internal O{sub 2}, and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O{sub 2} was lowered. Nitrogenase activity was stimulated by succinate but only at high external O{sub 2}. Oxygen uptake increased linearly with external O{sub 2} but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O{sub 2} concentration within detached nodules.

  1. Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform.

    PubMed

    Wilson, P E; Nyborg, A C; Watt, G D

    2001-07-24

    The Thorneley and Lowe kinetic model for nitrogenase catalysis was developed in the early to mid 1980s, and has been of value in accounting for many aspects of nitrogenase catalysis. It has also been of value by providing a model for predicting new catalytic behavior. Since its original publication, new results have been obtained and have been successfully incorporated into the model. However, the computer program used for nitrogenase simulations has not been generally available. Using kinetic schemes and assumptions previously outlined by Thorneley and Lowe, we report attempts to duplicate the original T&L kinetic simulation for Klebsiella pneumoniae nitrogenase catalysis using an updated simulation based on the MATHEMATICA programming format, which makes it more user-friendly and more readily available. Comparisons of our simulations with the original T&L simulations are generally in agreement, but in some cases serious discrepancy is observed. Possible reasons for the differences are discussed. In addition to duplicating the original T&L model, we report effects of updating it by including information that has come to light subsequent to its original publication.

  2. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.

    PubMed

    Brown, Katherine A; Harris, Derek F; Wilker, Molly B; Rasmussen, Andrew; Khadka, Nimesh; Hamby, Hayden; Keable, Stephen; Dukovic, Gordana; Peters, John W; Seefeldt, Lance C; King, Paul W

    2016-04-22

    The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3. Copyright © 2016, American Association for the Advancement of Science.

  3. Structures and Activities of the Elongator Complex and Its Cofactors.

    PubMed

    Kolaj-Robin, Olga; Séraphin, Bertrand

    2017-01-01

    Elongator is a highly conserved eukaryotic protein complex consisting of two sets of six Elp proteins, while homologues of its catalytic subunit Elp3 are found in all the kingdoms of life. Although it was originally described as a transcription elongation factor, cumulating evidence suggests that its primary function is catalyzing tRNA modifications. In humans, defects in Elongator subunits are associated with neurological disorders and cancer. Although further studies are still required, a clearer picture of the molecular mechanism of action of Elongator and its cofactors has started to emerge within recent years that have witnessed significant development in the field. In this review we summarize recent Elongator-related findings provided largely by crystal structures of several subunits of the complex, the electron microscopy structure of the entire yeast holoenzyme, as well as the structure of the Elongator cofactor complex Kti11/Kti13. © 2017 Elsevier Inc. All rights reserved.

  4. Enzymic synthesis and cofactor activity of 3'-pyrophosphocoenzyme A.

    PubMed Central

    Mukai, J I; Sy, J; Lipmann, F

    1983-01-01

    The 3'-pyrophosphate derivative of CoA was synthesized by using the excreted 5'-to-3' pyrophosphoryl-transferring enzyme from Streptomyces adephospholyticus and ATP as donor and dephospho-CoA as acceptor. Cofactor activity of this new coenzyme A derivative was tested with Clostridium kluyveri phosphotransacetylase and hog heart succinic thiokinase. With the phosphotransacetylase, 3'-pyrophospho-CoA was found to be twice as active as CoA whereas dephospho-CoA was inactive. However, succinic thiokinase utilized all three types of CoA equally well. Adenosine 5'-monophosphate 3'-pyrophosphate also was synthesized and used as an analog of adenosine 5'-monophosphate 3'-monophosphate in the dog liver's sulfotransferase-catalyzed sulfate transfer from p-nitrophenyl sulfate to phenol. In contrast to the pyrophospho derivative of coenzyme A, adenosine 5'-monophosphate 3'-pyrophosphate was inactive as a cofactor. PMID:6574458

  5. The chemical mechanism of nitrogenase: hydrogen tunneling and further aspects of the intramolecular mechanism for hydrogenation of eta(2)-N(2) on FeMo-co to NH(3).

    PubMed

    Dance, Ian

    2008-11-21

    The preceding paper (Dalton Trans., 2008, DOI: 10.1039/b806100a) describes the logical development of a chemical mechanism for the catalysis of hydrogenation of N(2) to 2NH(3) that occurs at the Fe(7)MoS(9)N(c)(homocitrate) cofactor (FeMo-co) of the enzyme nitrogenase. The mechanism uses a single replenishable path for serial supply of protons which become H atoms on FeMo-co, migrating to become S-H and Fe-H donors to N(2) and to the intermediates that follow. This chemical catalysis at FeMo-co is distinctly intramolecular: transition states and reaction profiles for the preferred 21 step pathway were presented. This paper describes a number of alternative intermediates and pathways that were considered in developing the mechanism. These results reveal further relevant principles of the reactivity of hydrogenated FeMo-co, and the reasons why these pathways are less likely to be part of the mechanism. The intramolecular character of the mechanism, and the relatively small distances over which H atoms transfer, lead to expectations of extensive quantum mechanical hydrogen tunneling as part of the catalytic rate enhancement. This possibility is supported by comparisons of reaction profiles with those for enzyme reactions for which tunneling is established.

  6. Copper is a Cofactor of the Formylglycine‐Generating Enzyme

    PubMed Central

    Knop, Matthias; Dang, Thanh Quy; Jeschke, Gunnar

    2016-01-01

    Abstract Formylglycine‐generating enzyme (FGE) is an O2‐utilizing oxidase that converts specific cysteine residues of client proteins to formylglycine. We show that CuI is an integral cofactor of this enzyme and binds with high affinity (K D=of 10−17  m) to a pair of active‐site cysteines. These findings establish FGE as a novel type of copper enzyme. PMID:27862795

  7. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    NASA Astrophysics Data System (ADS)

    Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-04-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  8. Coimmobilization of a Redox Enzyme and a Cofactor Regeneration System

    DTIC Science & Technology

    2006-07-01

    biocatalysis is widely used for redox reactions including asymmetric hydroxylations and epoxidations.2a Unfortunately, whole-cell systems are often...of purified redox enzymes in biocatalysis is limited by the cost of supplying stoichiometric amounts of cofactors for catalysis. An increasing...reduction of nitrobenzene to hydroxylaminobenzene (HAB) and has been successfully employed for whole-cell biocatalysis in o-aminophenol synthesis.4

  9. Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor.

    PubMed

    Amrani, L; Primus, J; Glatigny, A; Arcangeli, L; Scazzocchio, C; Finnerty, V

    2000-10-01

    The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.

  10. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  11. Organic cofactors in the metabolism of Dehalococcoides mccartyi strains

    PubMed Central

    Schipp, Christian J.; Marco-Urrea, Ernest; Kublik, Anja; Seifert, Jana; Adrian, Lorenz

    2013-01-01

    Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes. PMID:23479751

  12. The argon-induced decline in nitrogenase activity commences before the beginning of a decline in nodule oxygen uptake.

    PubMed

    Fischinger, Stephanie A; Schulze, Joachim

    2010-09-01

    Replacement of N(2) by argon in the air around nodules directs nitrogenase electron flow in its total onto H(+) resulting in increased nodule H(2) evolution (total nitrogenase activity (TNA)). However, argon application induces a so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with decreased nodule oxygen permeability. Consequently, TNA measurements tend to underestimate total nitrogenase activity. It is unclear whether the decline in oxygen diffusion into nodules induces the Ar-ID, or whether a decline in nitrogenase activity is followed by lower nodule O(2) uptake. The objective of the present work was to examine the time sequence of the decline in nodule H(2) evolution and O(2) uptake after argon application. In addition, the reliability of TNA values, taken as quickly as possible after the switch to Ar/O(2), was tested through comparative measurement of (15)N(2) uptake of the same plants. Short-term TNA measurements in an optimized gas exchange measurement system yielded reliable results, verified by parallel determination of (15)N(2) uptake. A five min application of Ar/O(2) was without effect on the subsequent H(2) evolution in ambient air. A parallel experiment on control plants revealed that a decrease in nodule oxygen uptake began several minutes after the onset of the decline in H(2) evolution. We conclude that the primary effect of the replacement of N(2) by argon differs from oxygen diffusion control. A gas exchange system allowing an immediate taking of TNA yields reliable results and does not disturb nodule activity. Gas exchange measurements provide a powerful tool for studying nodule physiology and should be combined with material from molecular studies.

  13. Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120 1

    PubMed Central

    Martín-Nieto, José; Herrero, Antonia; Flores, Enrique

    1991-01-01

    Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels. ImagesFigure 1Figure 2 PMID:16668475

  14. Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

    PubMed Central

    Hoffmann, Marie-Christine; Wagner, Eva; Langklotz, Sina; Pfänder, Yvonne; Hött, Sina; Bandow, Julia E.

    2015-01-01

    ABSTRACT Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed. IMPORTANCE Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression

  15. Expression of β-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra

    PubMed Central

    Legocki, Roman P.; Yun, Allen C.; Szalay, Aladar A.

    1984-01-01

    A 365-base-pair (bp) DNA fragment, containing the promoter region of the nitrogenase reductase (nifH) gene from stem Rhizobium BTAi1, has been isolated and sequenced. The transcription initiation sites were localized at positions 152 (major initiation) and 114 (minor initiation) nucleotides upstream of the translation initiation codon. The 200-bp nucleotide sequence upstream of the nifH structural gene shows substantial homology to the corresponding nifH regions of cowpea Rhizobium (100%), Parasponia Rhizobium (89%), and Rhizobium japonicum (88%). The nifH promoter region of stem Rhizobium BTAi1 was fused to the lacZ gene of Escherichia coli. The fusion and a 1.6-kilobase DNA specifying neomycin phosphotransferase were inserted into a 3,4-kilobase fragment of stem Rhizobium chromosome, and the resulting construct was placed on a mobilizable vector, pREV1000. Stem Rhizobium transconjugants resistant to kanamycin were found to contain the nifH promoter region-lacZ fusion linked to the neomycin phosphotransferase gene at the site of chromosomal homology. Analysis of the DNA from stable transconjugants showed integration of a single copy of these sequences into the chromosome by a double-reciprocal crossover event. The transconjugants formed nitrogen-fixing nodules, indicating that the insertion occurred in a “nonessential” region of the stem Rhizobium chromosome. Transconjugant strain BTAi1000 grows on β-galactosidase indicator plates under aerobic conditions as white colonies, whereas under microaerobic conditions (97% N2/3% O2), which derepress nitrogenase, the colonies turn blue within 15-24 hr. β-Galactosidase activity in derepressed cultures of BTAi1000 showed a 200-fold increase in comparison to the wild-type strain, whereas stem nodules formed by BTAi1000 exhibited 15- to 20-fold higher β-galactosidase values than wild-type nodules. Nitrogenase promoter-dependent expression of β-galactosidase in stem nodules was inhibited by fixed nitrogen, suggesting

  16. Constraints on texture zero and cofactor zero models for neutrino mass

    SciTech Connect

    Whisnant, K.; Liao, Jiajun; Marfatia, D.

    2014-06-24

    Imposing a texture or cofactor zero on the neutrino mass matrix reduces the number of independent parameters from nine to seven. Since five parameters have been measured, only two independent parameters would remain in such models. We find the allowed regions for single texture zero and single cofactor zero models. We also find strong similarities between single texture zero models with one mass hierarchy and single cofactor zero models with the opposite mass hierarchy. We show that this correspondence can be generalized to texture-zero and cofactor-zero models with the same homogeneous costraints on the elements and cofactors.

  17. Salinity effects on growth, photosynthetic parameters, and nitrogenase activity in estuarine planktonic cyanobacteria.

    PubMed

    Moisander, P H; McClinton, E; Paerl, H W

    2002-05-01

    Salinity has been suggested as being a controlling factor for blooms of N2-fixing cyanobacteria in estuaries. We tested the effect of salinity on the growth, N2 fixation, and photosynthetic activities of estuarine and freshwater isolates of heterocystous bloom-forming cyanobacteria. Anabaena aphanizomenoides and Anabaenopsis sp. were isolated from the Neuse River Estuary, North Carolina, and Cylindrospermopsis raciborskii from Lakes Dora and Griffin, central Florida. Salinity tolerance of these cyanobacteria was compared with that of two Nodularia strains from the Baltic Sea. We measured growth rates, N2 fixation (nitrogenase activity), and CO2 fixation at salinities between 0 and 20 g L(-1) NaCl. We also examined photosynthesis-irradiance relation-ships in response to salinity. Anabaenopsis maintained similar growth rates in the full range of salinities from 2 to 20 g L(-1) NaCl. Anabaena grew at up to 15 g L-', but the maximum salinity 20 g L(-1) NaCl was inhibitory. The upper limit for salinity tolerance of Cylindrospermopsis was 4 g L(-1) NaCl. Nodularia spp. maintained similar growth rates in the full range of salinities from 0 to 20 g L(-1) . Between 0 and 10 g L(-1), the growth rate of Nodularia spumigena was slower than that of the Neuse Estuary strains. In most strains, the sensitivity of nitrogenase activity and CO2 fixation to salinity appeared similar. Anabaenopsis, Anabaena, and the two Nodularia strains rapidly responded to NaCl by increasing their maximum photosynthetic rates (Pmn). Overall, both Neuse River Estuary and Baltic Sea strains showed an ability to acclimate to salt stress over short-(24 h) and long-term (several days to weeks) exposures. The study suggested that direct effect of salinity (as NaCl in these experiments) on cyanobacterial physiology does not alone explain the low frequency and magnitude of blooms of N2-fixing cyanobacteria in estuaries.

  18. Nitrogenase-mimic iron-containing chalcogels for photochemical reduction of dinitrogen to ammonia

    DOE PAGES

    Liu, Jian; Kelley, Matthew S.; Wu, Weiqiang; ...

    2016-05-02

    A nitrogenase-inspired biomimetic chalcogel system comprising double-cubane [Mo2Fe6S8(SPh)3] and single-cubane (Fe4S4) biomimetic clusters demonstrates photocatalytic N2 fixation and conversion to NH3 in ambient temperature and pressure conditions. Replacing the Fe4S4 clusters in this system with other inert ions such as Sb3+, Sn4+, Zn2+ also gave chalcogels that were photocatalytically active. Finally, molybdenum-free chalcogels containing only Fe4S4 clusters are also capable of accomplishing the N2 fixation reaction with even higher efficiency than their Mo2Fe6S8(SPh)3-containing counterparts. In this study, our results suggest that redox-active iron-sulfide–containing materials can activate the N2 molecule upon visible light excitation, which can be reduced all of themore » way to NH3 using protons and sacrificial electrons in aqueous solution. Evidently, whereas the Mo2Fe6S8(SPh)3 is capable of N2 fixation, Mo itself is not necessary to carry out this process. The initial binding of N2 with chalcogels under illumination was observed with in situ diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS). 15N2 isotope experiments confirm that the generated NH3 derives from N2. Density functional theory (DFT) electronic structure calculations suggest that the N2 binding is thermodynamically favorable only with the highly reduced active clusters. Finally, the results reported herein contribute to ongoing efforts of mimicking nitrogenase in fixing nitrogen and point to a promising path in developing catalysts for the reduction of N2 under ambient conditions.« less

  19. Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus

    PubMed Central

    McDonald, Ryan; Zhang, Fan; Watts, Joy E M; Schreier, Harold J

    2015-01-01

    The Amazonian catfish, Panaque nigrolineatus, consume large amounts of wood in their diets. The nitrogen-fixing community within the gastrointestinal (GI) tract of these catfish was found to include nifH phylotypes that are closely related to Clostridium sp., Alpha and Gammaproteobacteria, and sequences associated with GI tracts of lower termites. Fish fed a diet of sterilized palm wood were found to contain nifH messenger RNA within their GI tracts, displaying high sequence similarity to the nitrogen-fixing Bradyrhizobium group. Nitrogenase activity, measured by acetylene reduction assays, could be detected in freshly dissected GI tract material and also from anaerobic enrichment cultures propagated in nitrogen-free enrichment media; nifH sequences retrieved from these cultures were dominated by Klebsiella- and Clostridium-like sequences. Microscopic examination using catalyzed reporter deposition-enhanced immunofluorescence revealed high densities of nitrogenase-containing cells colonizing the woody digesta within the GI tract, as well as cells residing within the intestinal mucous layer. Our findings suggest that the P. nigrolineatus GI tract provides a suitable environment for nitrogen fixation that may facilitate production of reduced nitrogen by the resident microbial population under nitrogen limiting conditions. Whether this community is providing reduced nitrogen to the host in an active or passive manner and whether it is present in a permanent or transient relationship remains to be determined. The intake of a cellulose rich diet and the presence of a suitable environment for nitrogen fixation suggest that the GI tract microbial community may allow a unique trophic niche for P. nigrolineatus among fish. PMID:25909976

  20. Nitrogenase-mimic iron-containing chalcogels for photochemical reduction of dinitrogen to ammonia

    PubMed Central

    Liu, Jian; Kelley, Matthew S.; Wu, Weiqiang; Banerjee, Abhishek; Douvalis, Alexios P.; Wu, Jinsong; Zhang, Yongbo; Schatz, George C.; Kanatzidis, Mercouri G.

    2016-01-01

    A nitrogenase-inspired biomimetic chalcogel system comprising double-cubane [Mo2Fe6S8(SPh)3] and single-cubane (Fe4S4) biomimetic clusters demonstrates photocatalytic N2 fixation and conversion to NH3 in ambient temperature and pressure conditions. Replacing the Fe4S4 clusters in this system with other inert ions such as Sb3+, Sn4+, Zn2+ also gave chalcogels that were photocatalytically active. Finally, molybdenum-free chalcogels containing only Fe4S4 clusters are also capable of accomplishing the N2 fixation reaction with even higher efficiency than their Mo2Fe6S8(SPh)3-containing counterparts. Our results suggest that redox-active iron-sulfide–containing materials can activate the N2 molecule upon visible light excitation, which can be reduced all of the way to NH3 using protons and sacrificial electrons in aqueous solution. Evidently, whereas the Mo2Fe6S8(SPh)3 is capable of N2 fixation, Mo itself is not necessary to carry out this process. The initial binding of N2 with chalcogels under illumination was observed with in situ diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS). 15N2 isotope experiments confirm that the generated NH3 derives from N2. Density functional theory (DFT) electronic structure calculations suggest that the N2 binding is thermodynamically favorable only with the highly reduced active clusters. The results reported herein contribute to ongoing efforts of mimicking nitrogenase in fixing nitrogen and point to a promising path in developing catalysts for the reduction of N2 under ambient conditions. PMID:27140630

  1. Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus.

    PubMed

    McDonald, Ryan; Zhang, Fan; Watts, Joy E M; Schreier, Harold J

    2015-12-01

    The Amazonian catfish, Panaque nigrolineatus, consume large amounts of wood in their diets. The nitrogen-fixing community within the gastrointestinal (GI) tract of these catfish was found to include nifH phylotypes that are closely related to Clostridium sp., Alpha and Gammaproteobacteria, and sequences associated with GI tracts of lower termites. Fish fed a diet of sterilized palm wood were found to contain nifH messenger RNA within their GI tracts, displaying high sequence similarity to the nitrogen-fixing Bradyrhizobium group. Nitrogenase activity, measured by acetylene reduction assays, could be detected in freshly dissected GI tract material and also from anaerobic enrichment cultures propagated in nitrogen-free enrichment media; nifH sequences retrieved from these cultures were dominated by Klebsiella- and Clostridium-like sequences. Microscopic examination using catalyzed reporter deposition-enhanced immunofluorescence revealed high densities of nitrogenase-containing cells colonizing the woody digesta within the GI tract, as well as cells residing within the intestinal mucous layer. Our findings suggest that the P. nigrolineatus GI tract provides a suitable environment for nitrogen fixation that may facilitate production of reduced nitrogen by the resident microbial population under nitrogen limiting conditions. Whether this community is providing reduced nitrogen to the host in an active or passive manner and whether it is present in a permanent or transient relationship remains to be determined. The intake of a cellulose rich diet and the presence of a suitable environment for nitrogen fixation suggest that the GI tract microbial community may allow a unique trophic niche for P. nigrolineatus among fish.

  2. Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum.

    PubMed

    Meyer, J; Gaillard, J; Moulis, J M

    1988-08-09

    Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Coordinated Expression of fdxD and Molybdenum Nitrogenase Genes Promotes Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Oxygen

    PubMed Central

    Hoffmann, Marie-Christine; Müller, Alexandra; Fehringer, Maria; Pfänder, Yvonne; Narberhaus, Franz

    2014-01-01

    Rhodobacter capsulatus is able to grow with N2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression. PMID:24272776

  4. Design of dinuclear manganese cofactors for bacterial reaction centers.

    PubMed

    Olson, Tien L; Espiritu, Eduardo; Edwardraja, Selvakumar; Simmons, Chad R; Williams, JoAnn C; Ghirlanda, Giovanna; Allen, James P

    2016-05-01

    A compelling target for the design of electron transfer proteins with novel cofactors is to create a model for the oxygen-evolving complex, a Mn4Ca cluster, of photosystem II. A mononuclear Mn cofactor can be added to the bacterial reaction center, but the addition of multiple metal centers is constrained by the native protein architecture. Alternatively, metal centers can be incorporated into artificial proteins. Designs for the addition of dinuclear metal centers to four-helix bundles resulted in three artificial proteins with ligands for one, two, or three dinuclear metal centers able to bind Mn. The three-dimensional structure determined by X-ray crystallography of one of the Mn-proteins confirmed the design features and revealed details concerning coordination of the Mn center. Electron transfer between these artificial Mn-proteins and bacterial reaction centers was investigated using optical spectroscopy. After formation of a light-induced, charge-separated state, the experiments showed that the Mn-proteins can donate an electron to the oxidized bacteriochlorophyll dimer of modified reaction centers, with the Mn-proteins having additional metal centers being more effective at this electron transfer reaction. Modeling of the structure of the Mn-protein docked to the reaction center showed that the artificial protein likely binds on the periplasmic surface similarly to cytochrome c2, the natural secondary donor. Combining reaction centers with exogenous artificial proteins provides the opportunity to create ligands and investigate the influence of inhomogeneous protein environments on multinuclear redox-active metal centers. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  5. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    PubMed Central

    Giancaspero, Teresa A.; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M.; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD “chaperone.” The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells. PMID:25954742

  6. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis.

    PubMed

    Giancaspero, Teresa A; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina M; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

    2015-01-01

    The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  7. Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

    PubMed

    Vorholt, Julia A

    2002-10-01

    Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).

  8. NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

    PubMed

    Carugo, O; Argos, P

    1997-05-01

    The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.

  9. Biochemistry: role of PQQ as a mammalian enzyme cofactor?

    PubMed

    Felton, Leigh M; Anthony, Chris

    2005-02-03

    The announcement by Kasahara and Kato of a new redox-cofactor vitamin for mammals, pyrroloquinoline quinone (PQQ), was based on their claim that an enzyme, predicted to be involved in mouse lysine metabolism, is a PQQ-dependent dehydrogenase. However, this claim was dependent on a sequence analysis using databases that inappropriately label beta-propeller sequences as PQQ-binding motifs. What the evidence actually suggests is that the enzyme is an interesting novel protein that has a seven-bladed beta-propeller structure, but there is nothing to indicate that it is a PQQ-dependent dehydrogenase.

  10. Effect of high pN2 and high pD2 on NH3 production, H2 evolution, and HD formation by nitrogenases

    SciTech Connect

    Jensen, B.B.; Burris, R.H.

    1985-02-26

    We have investigated the effect of the partial pressure of N2 and D2 on HD formation, H2 evolution, and NH3 production by nitrogenase from Klebsiella pneumoniae and Clostridium pasteurianum. By using pressures up to 4 atm, we have been able to extend the concentration range of N2 and D2 in our investigations beyond that used in previous studies. The pN2 dependence of HD formation with constant pD2 ideally shows no HD formation under zero pN2, reaches a peak which depends on the pD2, and then decreases to zero at very high pN2. K. pneumoniae and C. pasteurianum nitrogenases differ in their Ki(D2) for nitrogen fixation. C. pasteurianum nitrogenase had the lower activity for formation of HD. With K. pneumoniae nitrogenase, D2 enhanced H2 evolution from 31% of the electron flux partitioned to H2 in the absence of D2 to 51% of the electron flux partitioned to H2 at 400 kPa of D2. With C. pasteurianum nitrogenase, the equivalent values were 33% and 48% of the total electron flux. Our results support previou findings on the mechanism for nitrogenase-catalyzed reductions proposed by W. W. Cleland.

  11. ENDOR/HYSCORE Studies of the Common Intermediate Trapped During Nitrogenase Reduction of N2H2, CH3N2H, and N2H4 Support an Alternating Reaction Pathway for N2 Reduction

    PubMed Central

    Lukoyanov, Dmitriy; Dikanov, Sergei A.; Yang, Zhi-Yong; Barney, Brett M.; Samoilova, Rimma I.; Narasimhulu, Kuppala V.; Dean, Dennis R.; Seefeldt, Lance C.; Hoffman, Brian M.

    2011-01-01

    Enzymatic N2 reduction proceeds along a reaction pathway comprised of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e−/H+ delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the ‘Distal’ (D) pathway, a single N of N2 is hydrogenated in three steps until the first NH3 is liberated, then the remaining nitrido-N is hydrogenated three more times to yield the second NH3. In the ‘Alternating’ (A) pathway, the two N’s instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH3 liberated only during the fifth step. A recent combination of X/Q-band EPR and 15N, 1,2H ENDOR measurements suggested that states trapped during turnover of the α-70Ala/α-195Gln MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [NxHy] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and 14/15N, 1,2H ENDOR/-HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N2H2, CH3N2H, and N2H4 in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N2H2, CH3N2H, and N2H4 via a common A reaction pathway, and that the same is true for N2 itself, with Fe ion(s) providing the site of reaction. PMID:21744838

  12. Multi-omic dynamics associate oxygenic photosynthesis with nitrogenase-mediated H2 production in Cyanothece sp. ATCC 51142

    DOE PAGES

    Bernstein, Hans C.; Charania, Moiz A.; McClure, Ryan S.; ...

    2015-11-03

    This study combines transcriptomic and proteomic profiling to provide new insights on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in the model cyanobacterium, Cyanothece sp. ATCC 51142. To date, the proposed mechanisms used to describe the energy metabolism processes that support H2 production in Cyanothece 51142 have assumed that ATP and reductant requirements are derived solely from glycogen oxidation and/or cyclic-electron flow around photosystem I. The results from this study present and test an alternative hypothesis by showing that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized withmore » nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the likely role of photocatalytic H2O oxidation as a major participating process.« less

  13. Cervical cancer: is herpes simplex virus type II a cofactor?

    PubMed Central

    Jones, C

    1995-01-01

    In many ways, cervical cancer behaves as a sexually transmitted disease. The major risk factors are multiple sexual partners and early onset of sexual activity. Although high-risk types of human papillomaviruses (HPV) play an important role in the development of nearly all cases of cervical cancer, other sexually transmitted infectious agents may be cofactors. Herpes simplex virus type 2 (HSV-2) is transmitted primarily by sexual contact and therefore has been implicated as a risk factor. Several independent studies suggest that HSV-2 infections correlate with a higher than normal incidence of cervical cancer. In contrast, other epidemiological studies have concluded that infection with HSV-2 is not a major risk factor. Two separate transforming domains have been identified within the HSV-2 genome, but continued viral gene expression apparently is not necessary for neoplastic transformation. HSV infections lead to unscheduled cellular DNA synthesis, chromosomal amplifications, and mutations. These observations suggest that HSV-2 is not a typical DNA tumor virus. It is hypothesized that persistent or abortive infections induce permanent genetic alterations that interfere with differentiation of cervical epithelium and subsequently induce abnormal proliferation. Thus, HSV-2 may be a cofactor in some but not all cases of cervical cancer. PMID:8665469

  14. Biochemistry of B12-cofactors in human metabolism.

    PubMed

    Kräutler, Bernhard

    2012-01-01

    Vitamin B12, the "antipernicious anaemia factor", is a crystallisable cobalt-complex, which belongs to a group of unique "complete" corrinoids, named cobalamins (Cbl). In humans, instead of the "vitamin", two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.

  15. Oxygen diffusion pathways in a cofactor-independent dioxygenase

    PubMed Central

    Di Russo, Natali V.; Condurso, Heather L.; Li, Kunhua; Bruner, Steven D.; Roitberg, Adrian E.

    2015-01-01

    Molecular oxygen plays an important role in a wide variety of enzymatic reactions. Through recent research efforts combining computational and experimental methods a new view of O2 diffusion is emerging, where specific channels guide O2 to the active site. The focus of this work is DpgC, a cofactor-independent oxygenase. Molecular dynamics simulations, together with mutagenesis experiments and xenon-binding data, reveal that O2 reaches the active site of this enzyme using three main pathways and four different access points. These pathways connect a series of dynamic hydrophobic pockets, concentrating O2 at a specific face of the enzyme substrate. Extensive molecular dynamics simulations provide information about which pathways are more frequently used. This data is consistent with the results of kinetic measurements on mutants and is difficult to obtain using computational cavity-location methods. Taken together, our results reveal that although DpgC is rare in its ability of activating O2 in the absence of cofactors or metals, the way O2 reaches the active site is similar to that reported for other O2-using proteins: multiple access channels are available, and the architecture of the pathway network can provide regio- and stereoselectivity. Our results point to the existence of common themes in O2 access that are conserved among very different types of proteins. PMID:26508997

  16. Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

    PubMed

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-04

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  17. Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat.

    PubMed

    Allen, G C; Grimm, D T; Elkan, G H

    1991-11-01

    Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 muM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 muM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 muM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min mg of protein and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and beta-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C(2)H(4)h mg (dry weight), and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C(2)H(4) h mg (dry weight). However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in "washout," proving that ORS571 can grow on N(2) and that there was not a nitrogen source in the medium that could substitute.

  18. Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yuichiro; Takashima, Yoshinori; Hashidzume, Akihito; Yamaguchi, Hiroyasu; Harada, Akira

    2015-11-01

    Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme-cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes.

  19. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    PubMed Central

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-01-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data. PMID:27418477

  20. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    NASA Astrophysics Data System (ADS)

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-07-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.

  1. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  2. Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel

    PubMed Central

    Kobayashi, Yuichiro; Takashima, Yoshinori; Hashidzume, Akihito; Yamaguchi, Hiroyasu; Harada, Akira

    2015-01-01

    Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme–cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes. PMID:26537172

  3. Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast

    PubMed Central

    López-Torrejón, Gema; Jiménez-Vicente, Emilio; Buesa, José María; Hernandez, Jose A.; Verma, Hemant K.; Rubio, Luis M.

    2016-01-01

    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. PMID:27126134

  4. Structural analysis of the genes encoding the molybdenum-iron protein of nitrogenase in the Parasponia rhizobium strain ANU289.

    PubMed Central

    Weinman, J J; Fellows, F F; Gresshoff, P M; Shine, J; Scott, K F

    1984-01-01

    The genes encoding the Molybdenum-Iron protein component of nitrogenase (nifD and nifK) have been identified and fully characterised in the Parasponia Rhizobium strain ANU289. The two genes are contiguous and are separated from the gene encoding the Fe-protein component of nitrogenase (nifH) by 21 kb of DNA. We present the entire DNA sequence of the nifD and nifK genes, thus completing the characterisation of the primary structure of the nitrogenase genes in this Rhizobium strain. Comparison of the sequence preceding the transcription initiation point of nifDK with that preceding nifH reveals a consensus promoter sequence 5'-PyTGGCAPyG-4 bp-TTGC(T/A)-10 bp-3'. This consensus promoter is found preceding nif genes in both fast-growing and slow-growing Rhizobium strains and shows a structural similarity to that preceding the coordinately-regulated nif operons in the asymbiotic organism Klebsiella pneumoniae. Images PMID:6095197

  5. Foliar application of pyraclostrobin fungicide enhances the growth, rhizobial-nodule formation and nitrogenase activity in soybean (var. JS-335).

    PubMed

    Joshi, Juhie; Sharma, Sonika; Guruprasad, K N

    2014-09-01

    A field study was conducted to investigate the impact of the fungicide pyraclostrobin (F500 - Headline®; a.i. 20%) on the activity of nitrogenase in soybean (var. JS-335). Pyraclostrobin (F500) was applied on the leaves of soybean plants at 10 and 20 days after emergence (DAE) of seedlings at concentrations ranging from 0.05% to 1%. Leghemoglobin (Lb) content and nitrogenase activity in root nodules were analyzed at 45(th)day after emergence of seedlings indicated a remarkable increase in Lb content and enhanced activity of nitrogenase in the root nodules of pyraclostrobin treated plants. The fungicide also enhanced the number of nodules along with weight of nodules, root biomass and growth of shoot and leaves. Enhanced nitrogen fixation in the root nodules by pyraclostrobin improves the growth of the plant in soybean before flowering and pod formation which ultimately resulted in yield and yield attributes. These results suggest that pyraclostrobin (F500) can be successfully employed as a foliar spray under field conditions to enhance the growth, nitrogen assimilation and hence yield of soybean.

  6. The stereochemistry and dynamics of the introduction of hydrogen atoms onto FeMo-co, the active site of nitrogenase.

    PubMed

    Dance, Ian

    2013-11-18

    The catalyzed hydrogenations effected at the active site FeMo-co of nitrogenase have been proposed to involve serial supply of the required multiple protons along a proton wire terminating at sulfur atom S3B of FeMo-co. In conjunction with serial electron transfer to FeMo-co, these protons become H atoms, and then are able to migrate from S3B to other Fe and S atoms of FeMo-co, and to transfer to bound substrate and intermediates. This general model, which can account for all reactions of nitrogenase, involves a preparatory stage in which each incoming H atom is required to move from the proton delivery side of S3B to the opposite migration side of S3B. This report examines the mechanism of this reconfiguration of S3B-H, finding four stable configurations in which S3B-H has pyramidal-trigonal coordination, with one elongated Fe-S3B interaction. The transition states and energies for reconfiguration are described. Pseudotetrahedral four coordination and planar-trigonal coordination for S3B-H are less stable than pyramidal-trigonal coordination. Results are presented for FeMo-co with one, two, three, and four H atoms (the E1H1, E2H2, E3H3, and E4H4 Thorneley-Lowe stages), and the general principles are defined, for application in the various chemical mechanisms of nitrogenase.

  7. Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast.

    PubMed

    López-Torrejón, Gema; Jiménez-Vicente, Emilio; Buesa, José María; Hernandez, Jose A; Verma, Hemant K; Rubio, Luis M

    2016-04-29

    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.

  8. Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism

    PubMed Central

    Boyd, Eric S.; Costas, Amaya M. Garcia; Hamilton, Trinity L.; Mus, Florence

    2015-01-01

    ABSTRACT Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. IMPORTANCE Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and

  9. Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism.

    PubMed

    Boyd, Eric S; Costas, Amaya M Garcia; Hamilton, Trinity L; Mus, Florence; Peters, John W

    2015-05-01

    Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and later diversified into

  10. Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

    PubMed Central

    Madden, M S; Kindon, N D; Ludden, P W; Shah, V K

    1990-01-01

    In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analogs allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (1R,2S)-threo- and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV- mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties of dinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of -isocitrate and shows strong CO inhibition of substrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures of these analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate

  11. Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1

    SciTech Connect

    Zhang, Aihua; Li, Chia-Wei; Chen, J. Don . E-mail: chenjd@umdnj.edu

    2007-07-13

    The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a p160 coactivator-interacting protein that may inhibit transcriptional activity of nuclear receptors. Here, we have characterized the transcriptional regulatory domains of ANCO-1. Two intrinsic repression domains (RD) were identified: an N-terminal RD1 at residues 318-611 and a C-terminal RD2 at 2369-2663. ANCO-1 also contains an activation domain (AD) capable of stimulating transcription in both mammalian and yeast cells. The minimal AD was delimited to a 70-amino acid region at residues 2076-2145. Overall, full-length ANCO-1 exhibited transcriptional repressor activity, suggesting that RD domains may suppress the AD activity. We further demonstrated that ANCO-1 silencing by siRNA enhanced progesterone receptor-mediated transcription. Together, these results indicate that the transcriptional potential of ANCO-1 may be modulated by a combination of repression and activation signals.

  12. Theoretical estimation of redox potential of biological quinone cofactors.

    PubMed

    Gillet, Natacha; Lévy, Bernard; Moliner, Vicent; Demachy, Isabelle; de la Lande, Aurélien

    2017-07-05

    Redox potentials are essential to understand biological cofactor reactivity and to predict their behavior in biological media. Experimental determination of redox potential in biological system is often difficult due to complexity of biological media but computational approaches can be used to estimate them. Nevertheless, the quality of the computational methodology remains a key issue to validate the results. Instead of looking to the best absolute results, we present here the calibration of theoretical redox potential for quinone derivatives in water coupling QM + MM or QM/MM scheme. Our approach allows using low computational cost theoretical level, ideal for long simulations in biological systems, and determination of the uncertainties linked to the calculations. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Dinitrogen and Cyanide Fixation by Methane Seep Microorganisms Revealed by FISH- SIMS And Implications for AOM Productivity and Nitrogenase Evolution

    NASA Astrophysics Data System (ADS)

    Dekas, A.; Orphan, V.

    2008-12-01

    The anaerobic oxidation of methane (AOM), mediated by methane oxidizing archaea (ANME) and sulfate reducing bacterial symbionts (SRB), minimizes the flux of methane from marine sediment to the overlying water column. Understanding the factors determining AOM productivity, and particularly the rates of methane catabolism and anabolism, is of interest to both modern and ancient investigations of climate and bulk carbon isotopic change. It has been hypothesized that nitrogen availability in methane seeps is temporally variable, and that the seep biomass may be at least partially nitrogen limited. The recent finding of nif genes, those necessary for the production of nitrogenase, in enrichments of ANME and SRB consortia suggested that the organisms mediating AOM have the potential to fix dinitrogen. In the present study we incubated methane seep sediment with nitrogen-deplete artificial marine media and a headspace of methane (CH4) and either 15N-labeled dinitrogen (15N2), cyanide (C15N-), or ammonia (15NH3) in order to (1) test the ability of these currently unculturable microorganisms to fix nitrogen and other triple bonded substrates, (2) investigate which AOM partner was responsible for the fixation, (3) compare growth rates on different nitrogen sources, and (4) characterize the phylogeny of these methane seep-associated nitrogenases. Fluorescence in situ hybridization coupled to nano-scale Secondary Ion Mass Spectroscopy imaging (FISH-SIMS) revealed incorporation of 15N into ANME and SRB biomass of up to 0.06 15N fractional abundance in the 15N2 incubation, and up to 0.02 in the C15N- incubation, after 6 and 4 months, respectively. This represents a nearly ten-fold enrichment of 15N compared to the measured natural 15N fractional abundance (0.0036). The NanoSIMS ion images of ANME/SRB aggregates from 15N2 incubations show evidence for 15N enrichment in both partners with the highest incorporation of 15N within the methanotrophic ANME cells. Cyanide incubations

  14. Nitrogenase activity and nifH expression in a marine intertidal microbial mat.

    PubMed

    Steppe, T F; Paerl, H W

    2005-02-01

    N(2) fixation, diazotrophic community composition, and organisms actively expressing genes for N(2) fixation were examined over at 3-year period (1997-1999) for intertidal microbial mats on a sand flat located in the Rachel Carson National Estuarine Research Reserve (RCNERR) (Beaufort, NC, USA). Specifically, diel variations of N(2) fixation in the mats from the RCNERR were examined. Three distinct diel patterns of nitrogenase activity (NA) were observed. NA responses to short-term inhibitions of photosynthesis corresponded to one of the three patterns. High rates of NA were observed during peak O(2) production periods for diel experiments during summer months. Different types of NA diel variations correspond to different stages of mat development. Chloramphenicol treatments indicated that the mechanism of protein synthesis supporting NA changed throughout the day. Analysis of mat DNA and RNA gave further evidence suggesting that in addition to cyanobacteria, other functional groups were responsible for the NA observed in the RCNERR mats. The role of microbial diversity in the N(2) fixation dynamics of these mats is discussed.

  15. Incidence of novel and potentially archaeal nitrogenase genes in the deep Northeast Pacific Ocean.

    PubMed

    Mehta, Mausmi P; Huber, Julie A; Baross, John A

    2005-10-01

    Archaea have been detected throughout the oceanic water column and are quantitatively important members of picoplankton in the deep ocean. Two common groups, group I Crenarchaeota and group II Euryarchaeota, are consistently detected in warm hydrothermal fluid and are assumed to have been drawn into the subseafloor, mixed with hydrothermal fluid and then expelled. However, because they remain resistant to cultivation, very little is known about their physiology. Here we show that cold deep-seawater from the axial valley of Endeavour Segment on the Juan de Fuca Ridge contains not only groups I and II archaea as expected, but also unique potentially archaeal nitrogenase (nifH) genes, which are required for nitrogen fixation. These nifH genes are phylogenetically distinct and have dissimilar G+C content compared with those of hydrothermal vent archaea, suggesting that they belong to non-thermophilic deep-sea archaea. Furthermore, this sample did not contain mcrA genes, which are present in methanogens, the only known archaeal nitrogen fixers. These nifH genes were not detected in upper water column samples, or in a deep-seawater sample 100 km away from the spreading axis of the Juan de Fuca Ridge. We propose that these unique nifH genes may be localized to archaea that circulate through the nitrogen-poor subseafloor at the mid-ocean ridge as part of their life cycle.

  16. Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite.

    PubMed Central

    Thorneley, R N; Yates, M G; Lowe, D J

    1976-01-01

    The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover. Images Fig. 1. Fig. 4. PMID:180978

  17. SQUID measurement of metalloprotein magnetization. New methods applied to the nitrogenase proteins.

    PubMed Central

    Day, E P; Kent, T A; Lindahl, P A; Münck, E; Orme-Johnson, W H; Roder, H; Roy, A

    1987-01-01

    New techniques have been developed to exploit the sensitivity of a commercial SQUID susceptometer in the study of the magnetization of metalloproteins. Previous studies have ignored both the slow relaxation (hours) of spin I = 1/2 nuclei and residual ferromagnetic impurities in sample holders. These potential sources of noise were at or below the sensitivity of previous instruments. With these noise sources under control, one can now decrease the protein concentration by a factor of ten. In addition careful characterization of the frozen magnetization sample, including the use of a multi-instrument holder for combined study of the magnetization sample with Mössbauer spectroscopy, is required for reliable interpretation of the data in the face of paramagnetic impurities common to metalloprotein samples. Many previous magnetic studies of metalloproteins have been carried out in the Curie region. Saturation magnetization studies down to 1.8 K and up to 5 T can determine zero-field splitting parameters in addition to the spin and exchange coupling parameters measured in previous studies at lower fields and higher temperatures. Applications of these techniques to the study of the nitrogenase proteins of Azotobacter vinelandii are presented as examples. PMID:3480761

  18. Wheat straw degradation and production of alternative substrates for nitrogenase of Rhodobacter sphaeroides.

    PubMed

    Dziga, Dariusz; Jagiełło-Flasińska, Dominika

    2015-01-01

    Cellulose is a major component of plant biomass and could be applied in the production of biofuels, especially bioethanol. An alternative approach is production of a clean fuel - hydrogen from cellulosic biomass. In this paper an innovatory model of cellulosic waste degradation has been proposed to verify the possibility of utilization of cellulose derivatives by purple non-sulfur bacteria. The concept is based on a two-step process of wheat straw conversion by bacteria in order to obtain an organic acid mixture. In the next stage such products are consumed by Rhodobacter sphaeroides, the known producer of hydrogen. It has been documented that Cellulomonas uda expresses cellulolytic activity in the presence of wheat straw as an only source of carbon. R. sphaeroides applied in this research can effectively consume organic acids released from straw by C. uda and Lactobacillus rhamnosus and is able to grow in the presence of these substrates. Additionally, an increased nitrogenase activity of R. sphaeroides has been indicated when bacteria were cultivated in the presence of cellulose derivatives which suggests that hydrogen production occurs.

  19. Nitrogenase expression in estuarine bacterioplankton influenced by organic carbon and availability of oxygen.

    PubMed

    Severin, Ina; Bentzon-Tilia, Mikkel; Moisander, Pia H; Riemann, Lasse

    2015-07-01

    The genetic capacity to fix gaseous nitrogen (N) is distributed among diverse diazotrophs belonging to the Bacteria and Archaea. However, only a subset of the putative diazotrophs present actively fix N at any given time in the environment. We experimentally tested whether the availability of carbon and inhibition by oxygen constrain N fixation by diazotrophs in coastal seawater. The goal was to test whether by alleviating these constraints an increased overlap between nitrogenase (nifH)-gene-carrying and -expressing organisms could be achieved. We incubated water from a eutrophic but N-limited fjord in Denmark under high-carbon/low-oxygen conditions and determined bacterial growth and production, diazotrophic community composition (Illumina nifH amplicon sequencing), and nifH gene abundance and expression [quantitative PCR (qPCR) and quantitative reverse transcriptase PCR (qRT-PCR)]. Bacterial abundances and production increased under high-carbon/low-oxygen conditions as did the similarity between present and active diazotrophic communities. This was caused by the loss of specific abundant yet non-active gammaproteobacterial phylotypes and increased expression by others. The prominent active gamma- and epsilonproteobacterial diazotrophs did not, however, respond to these conditions in a uniform way, highlighting the difficulty to assess how a change in environmental conditions may affect a diverse indigenous diazotrophic community. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Ammonia formation by a thiolate-bridged diiron amide complex as a nitrogenase mimic

    NASA Astrophysics Data System (ADS)

    Li, Yang; Li, Ying; Wang, Baomin; Luo, Yi; Yang, Dawei; Tong, Peng; Zhao, Jinfeng; Luo, Lun; Zhou, Yuhan; Chen, Si; Cheng, Fang; Qu, Jingping

    2013-04-01

    Although nitrogenase enzymes routinely convert molecular nitrogen into ammonia under ambient temperature and pressure, this reaction is currently carried out industrially using the Haber-Bosch process, which requires extreme temperatures and pressures to activate dinitrogen. Biological fixation occurs through dinitrogen and reduced NxHy species at multi-iron centres of compounds bearing sulfur ligands, but it is difficult to elucidate the mechanistic details and to obtain stable model intermediate complexes for further investigation. Metal-based synthetic models have been applied to reveal partial details, although most models involve a mononuclear system. Here, we report a diiron complex bridged by a bidentate thiolate ligand that can accommodate HN=NH. Following reductions and protonations, HN=NH is converted to NH3 through pivotal intermediate complexes bridged by N2H3- and NH2- species. Notably, the final ammonia release was effected with water as the proton source. Density functional theory calculations were carried out, and a pathway of biological nitrogen fixation is proposed.

  1. Epiphytic cyanobacteria of the seagrass Cymodocea rotundata: diversity, diel nifH expression and nitrogenase activity.

    PubMed

    Hamisi, Mariam; Díez, Beatriz; Lyimo, Thomas; Ininbergs, Karolina; Bergman, Birgitta

    2013-06-01

    Seagrasses are photoautotrophic, ecologically important components of many globally widespread coastal ecosystems, in which combined nitrogen may limit their production. We examined the biodiversity and diazotrophic capacity of microbial epiphytes associated with the phyllosphere of the seagrass Cymodocea rotundata of the Western Indian Ocean. Light microscopy, 16S rRNA and nifH gene analysis revealed the dominance of cyanobacteria in the epiphytic microbial community. Most phylotypes were related to free-living uncultured benthic cyanobacteria, while some to cyanobacterial endosymbionts of marine diatoms. Novel and potentially diazotrophic species, some of known pantropical distribution, were also discovered. Significant diel nitrogenase activities (acetylene reduction assay) were recorded (up to 358 ± 232 nmol C2H4 g(-1) of seagrass FW h(-1)). The nifH gene expression patterns showed that heterocystous phylotypes may be the dominant diazotrophs during the day and non-heterocystous at night. These data show that C. rotundata is colonized by diverse diazotrophic cyanobacteria species and suggest that these may be beneficial partners of seagrasses in nitrogen-depleted waters. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  2. Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates.

    PubMed

    Darnajoux, Romain; Zhang, Xinning; McRose, Darcy L; Miadlikowska, Jolanta; Lutzoni, François; Kraepiel, Anne M L; Bellenger, Jean-Philippe

    2017-01-01

    Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

  3. EPR monitored redox titration of the cofactors of Saccharomyces cerevisiae Nar1.

    PubMed

    Hagedoorn, Peter-Leon; van der Weel, Laura; Hagen, Wilfred R

    2014-11-26

    Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state. The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor. A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

  4. EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

    PubMed Central

    Hagedoorn, Peter-Leon; van der Weel, Laura; Hagen, Wilfred R.

    2014-01-01

    Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state. The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor. A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor. PMID:25490157

  5. The non-enzymatic reduction of azo dyes by flavin and nicotinamide cofactors under varying conditions.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2013-10-01

    Azo dyes are ubiquitous in products and often become environmental pollutants due to their anthropogenic nature. Azoreductases are enzymes which are present within many bacteria and are capable of breaking down the azo dyes via reduction of the azo bond. Often, though, carcinogenic aromatic amines are formed as metabolites and are of concern to humans. Azoreductases function via an oxidation-reduction reaction and require cofactors (a nicotinamide cofactor and sometimes a flavin cofactor) to perform their function. Non-enzymatic reduction of azo dyes in the absence of an azoreductase enzyme has been suggested in previous studies, but has never been studied in detail in terms of varying cofactor combinations, different oxygen states or pHs, nor has the enzymatic reduction been compared to azoreduction in terms of dye reduction or metabolites produced, which was the aim of this study. Reduction of azo dyes by different cofactor combinations was found to occur under both aerobic and anaerobic conditions and under physiologically-relevant pHs to produce the same metabolites as an azoreductase. Our results show that, in some cases, the non-enzymatic reduction by the cofactors was found to be equal to that seen with the azoreductase, suggesting that all dye reduction in these cases is due to the cofactors themselves. This study details the importance of the use of a cofactor-only control when studying azoreductase enzymes.

  6. Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor

    SciTech Connect

    Hoover, T.R.; Imperial, J.; Liang, J.; Ludden, P.W.; Shah, V.K.

    1988-05-17

    The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C/sub 2/H/sub 2/ reduction assays and the lack of /sup 99/Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of /sup 99/Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C/sub 2/H/sub 2/ or N/sub 2/. Citrate also promoted the incorporation of /sup 99/Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C/sub 2/H/sub 2/ effectively but not N/sub 2/. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV/sup -/ dinitrogenase. The authors also observed low rates of HD formation from NifV/sup -/ dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. They propose that in vivo NifV/sup -/ mutants utilize citrate for FeMo-co synthesis.

  7. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    PubMed Central

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-01-01

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch. PMID:23776225

  8. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

    PubMed

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K B; Snow, Christopher D; Brustad, Eric M; McIntosh, John A; Meinhold, Peter; Zhang, Liang; Arnold, Frances H

    2013-07-02

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  9. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    SciTech Connect

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  10. Co‐immobilized Phosphorylated Cofactors and Enzymes as Self‐Sufficient Heterogeneous Biocatalysts for Chemical Processes

    PubMed Central

    Velasco‐Lozano, Susana; Benítez‐Mateos, Ana I.

    2016-01-01

    Abstract Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD+, and NAD+) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations. PMID:28000978

  11. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  12. Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors.

    PubMed

    Eekels, Julia J M; Geerts, Dirk; Jeeninga, Rienk E; Berkhout, Ben

    2011-01-01

    In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.

  13. Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

    PubMed Central

    Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

    2014-01-01

    A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP+ and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically-modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin-peroxide intermediate in a catalytically-competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multi-cofactor active site, which has implications for enzyme design based on cofactor engineering approaches. PMID:24443704

  14. What's in a covalent bond? On the role and formation of covalently bound flavin cofactors.

    PubMed

    Heuts, Dominic P H M; Scrutton, Nigel S; McIntire, William S; Fraaije, Marco W

    2009-07-01

    Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin-protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein-flavin bonds. These endeavors have revealed that covalent flavinylation is a post-translational and self-catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation.

  15. Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes.

    PubMed

    Velasco-Lozano, Susana; Benítez-Mateos, Ana I; López-Gallego, Fernando

    2017-01-16

    Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD(+) , and NAD(+) ) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD(+) -dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

  16. Cofactors in allergic reactions to food: physical exercise and alcohol are the most important

    PubMed Central

    van Os‐Medendorp, Harmieke; Kruizinga, Astrid G.; Blom, W. Marty; Houben, Geert F.; Knulst, André C.

    2016-01-01

    Abstract Introduction Involvement of cofactors, like physical exercise, alcohol consumption and use of several types of medication, are associated with more severe food allergic symptoms. However, there is limited evidence on how often cofactors play a role in food allergic reactions. The study aimed to get more insight into the frequency of exposure to cofactors and how often cofactors are associated with more severe symptoms in food allergic patients. Methods A questionnaire was completed by patients visiting the Allergology outpatient clinic. Patients with food allergy were included. Outcome measures were the frequency of medication use of medication groups that might act as cofactor and the frequency that physical exercise, alcohol consumption and use of analgesics are associated with more severe food allergic symptoms. Results Four hundred ninety‐six patients were included in the study. The frequency with which patients used one or more types of medication that might act as cofactors was 7.7%: antacids/acid neutralizing medication (5%), NSAIDs (2%), beta blockers (0.6%), angiotensin‐converting enzyme inhibitors (0.6%), and angiotensin receptor blockers (0.2%). Of all patients, 13% reported more severe symptoms to food after involvement of one or more of the cofactors: physical exercise (10%), alcohol consumption (5%), and use of analgesics (0.6%). Sixty‐five percent did not know if these cofactors caused more severe symptoms; 22% reported that these cofactors had no effect. Conclusions Only a small percentage of patients (7.7%) used medication that might aggravate food allergic reactions. Physical exercise and alcohol consumption were the most frequently reported cofactors, but occurring still in only 10% or less. PMID:27980774

  17. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

    DOE PAGES

    Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.; ...

    2015-05-04

    The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product ofmore » in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, in this paper we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. In conclusion, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.« less

  18. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

    SciTech Connect

    Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.; Yokoyama, Kenichi

    2015-05-04

    The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, in this paper we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. In conclusion, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

  19. Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins

    PubMed Central

    Eady, R. R.; Smith, B. E.; Cook, K. A.; Postgate, J. R.

    1972-01-01

    1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s020,w=11.0S and D020,w=4.94×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H2 evolution, 380 for N2 reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s020,w=4.8S and D020,w=5.55×10−7cm2/s. The specific activities (nmol of product formed

  20. Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

    PubMed Central

    Kern, Monika; Klipp, Werner; Klemme, Jobst-Heinrich

    1994-01-01

    Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H2 photoproduction. However, a mutation in a structural hup gene did not result in maximum H2 production rates, indicating that the capacity to recycle H2 was not completely lost. Highest H2 production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H2 recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA. Images PMID:16349271

  1. Circular dichroism and magnetic circular dichroism of reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

    PubMed

    Stephens, P J; McKenna, C E; McKenna, M C; Nguyen, H T; Devlin, F

    1981-05-12

    Studies of the circular dichroism (CD) and magnetic circular dichroism (MCD) of the dithionite-reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase (Av1) are reported. CD and MCD are measurable at room temperature across a wide spectral range, from the near-UV to the near-IR. The visible-near-UV CD is insignificantly affected by moderate variations in pH, temperature, ionic strength, and buffer, providing evidence against conformational change in the range studied. Mg2+ and ATP also cause no observable change in the visible-near-UV CD. Both CD and MCD in the visible-near-UV are unaffected by 30% inactivation by O2. However, the CD and MCD spectra of uncrystallized Av1 differ very significantly from those of crystallized Av1; in particular, the MCD spectrum is very sensitive to the presence of heme impurities. The identicality in both CD and MCD spectra of the reduced molybdenum-iron proteins from Azotobacter vinelandii and Klebsiella pneumoniae shows that these proteins contain metal clusters, identical in number, structure, and protein environment. While the absorption, CD, and MCD spectra of reduced Av1 are typical in many respects of simpler iron-sulfur proteins and are most similar to the [Fe4S4(SR)4]3- clusters found in reduced bacterial ferredoxins, significant differences exist. It is concluded, therefore, that the clusters present are not identical with those previously characterized, a conclusion earlier arrived at from electron paramagnetic resonance, Mössbauer, and EXAFS spectroscopies.

  2. Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment.

    PubMed

    Hewson, Ian; Moisander, Pia H; Morrison, Amanda E; Zehr, Jonathan P

    2007-05-01

    We investigated diazotrophic bacterioplankton assemblage composition in the Heron Reef lagoon (Great Barrier Reef, Australia) using culture-independent techniques targeting the nifH fragment of the nitrogenase gene. Seawater was collected at 3 h intervals over a period of 72 h (i.e. over diel as well as tidal cycles). An incubation experiment was also conducted to assess the impact of phosphate (PO(4)3*) availability on nifH expression patterns. DNA-based nifH libraries contained primarily sequences that were most similar to nifH from sediment, microbial mat and surface-associated microorganisms, with a few sequences that clustered with typical open ocean phylotypes. In contrast to genomic DNA sequences, libraries prepared from gene transcripts (mRNA amplified by reverse transcription-polymerase chain reaction) were entirely cyanobacterial and contained phylotypes similar to those observed in open ocean plankton. The abundance of Trichodesmium and two uncultured cyanobacterial phylotypes from previous studies (group A and group B) were studied by quantitative-polymerase chain reaction in the lagoon samples. These were detected as transcripts, but were not detected in genomic DNA. The gene transcript abundance of these phylotypes demonstrated variability over several diel cycles. The PO(4)3* enrichment experiment had a clearer pattern of gene expression over diel cycles than the lagoon sampling, however PO(4)3* additions did not result in enhanced transcript abundance relative to control incubations. The results suggest that a number of diazotrophs in bacterioplankton of the reef lagoon may originate from sediment, coral or beachrock surfaces, sloughing into plankton with the flooding tide. The presence of typical open ocean phylotype transcripts in lagoon bacterioplankton may indicate that they are an important component of the N cycle of the coral reef.

  3. Temporal Patterns of Nitrogenase Gene (nifH) Expression in the Oligotrophic North Pacific Ocean

    PubMed Central

    Church, Matthew J.; Short, Cindy M.; Jenkins, Bethany D.; Karl, David M.; Zehr, Jonathan P.

    2005-01-01

    Dinitrogen (N2)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity. In this study, we examined the presence and expression of specific planktonic nitrogenase genes (nifH) in the upper ocean (0 to 175 m) at Station ALOHA in the oligotrophic North Pacific Ocean. Clone libraries constructed from reverse-transcribed PCR-amplified mRNA revealed six unique phylotypes. Five of the nifH phylotypes grouped with sequences from unicellular and filamentous cyanobacteria, and one of the phylotypes clustered with γ-proteobacteria. The cyanobacterial nifH phylotypes retrieved included two sequence types that phylogenetically grouped with unicellular cyanobacteria (termed groups A and B), several sequences closely related (97 to 99%) to Trichodesmium spp. and Katagnymene spiralis, and two previously unreported phylotypes clustering with heterocyst-forming nifH cyanobacteria. Temporal patterns of nifH expression were evaluated using reverse-transcribed quantitative PCR amplification of nifH gene transcripts. The filamentous and presumed unicellular group A cyanobacterial phylotypes exhibited elevated nifH transcription during the day, while members of the group B (closely related to Crocosphaera watsonii) unicellular phylotype displayed greater nifH transcription at night. In situ nifH expression by all of the cyanobacterial phylotypes exhibited pronounced diel periodicity. The γ-proteobacterial phylotype had low transcript abundance and did not exhibit a clear diurnal periodicity in nifH expression. The temporal separation of nifH expression by the various phylotypes suggests that open ocean diazotrophic cyanobacteria have unique in situ physiological responses to daily fluctuations of light in the upper ocean. PMID:16151126

  4. Nitrogenase gene amplicons from global marine surface waters are dominated by genes of non-cyanobacteria.

    PubMed

    Farnelid, Hanna; Andersson, Anders F; Bertilsson, Stefan; Al-Soud, Waleed Abu; Hansen, Lars H; Sørensen, Søren; Steward, Grieg F; Hagström, Åke; Riemann, Lasse

    2011-04-29

    Cyanobacteria are thought to be the main N(2)-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2) fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

  5. Similar Diversity of Alphaproteobacteria and Nitrogenase Gene Amplicons on Two Related Sphagnum Mosses

    PubMed Central

    Bragina, Anastasia; Maier, Stefanie; Berg, Christian; Müller, Henry; Chobot, Vladimir; Hadacek, Franz; Berg, Gabriele

    2012-01-01

    Sphagnum mosses represent a main vegetation component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand the extend of host specificity and impact of environment, Sphagnum fallax and Sphagnum angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-single-strand conformation polymorphism of 16S rRNA and nitrogenase-encoding (nifH) genes were highly similar for both Sphagnum species. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH) coupled with confocal laser scanning microscopy (CLSM): Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila, and Phenylobacterium as major genera for both mosses. Nitrogen fixation is an important function of Sphagnum-associated bacteria, which is fulfilled by microbial communities of Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g., Cyanobacteria and Deltaproteobacteria. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological, and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities. PMID:22294982

  6. Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the alpha-subunit of nitrogenase MoFe protein.

    PubMed Central

    Ioannidis, I; Buck, M

    1987-01-01

    The nucleotide sequence of the Klebsiella pneumoniae nifD gene is presented and together with the accompanying paper [Holland, Zilberstein, Zamir & Sussman (1987) Biochem. J. 247, 277-285] completes the sequence of the nifHDK genes encoding the nitrogenase polypeptides. The K. pneumoniae nifD gene encodes the 483-amino acid-residue nitrogenase alpha-subunit polypeptide of Mr 54156. The alpha-subunit has five strongly conserved cysteine residues at positions 63, 89, 155, 184 and 275, some occurring in a region showing both primary sequence and potential structural homology to the K. pneumoniae nitrogenase beta-subunit. A comparison with six other alpha-subunit amino acid sequences has been made, which indicates a number of potentially important domains within alpha-subunits. PMID:3322262

  7. The uptake hydrogenase in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain PCC 7822 protects nitrogenase from oxygen toxicity.

    PubMed

    Zhang, Xiaohui; Sherman, Debra M; Sherman, Louis A

    2014-02-01

    Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase.

  8. The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

    PubMed Central

    Zhang, Xiaohui; Sherman, Debra M.

    2014-01-01

    Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase. PMID:24317398

  9. Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium.

    PubMed Central

    MacNeil, D; Brill, W J

    1980-01-01

    Four Nif+ revertants from strains with polar insertions in nifL, were insensitive to ammonium and amino acid repression of nitrogenase synthesis. These strains have mutations located in or near the nifL region. The derepressed phenotype was dominant in a merodiploid containing a nif+ plasmid. These nif regulatory mutations also suppressed the Nif- phenotype of Gln- strains. Thus, regulation by fixed nitrogen (possible via glutamine synthetase) occurs on the nifLA operon but not on the other six nif operons. PMID:7000753

  10. Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat

    PubMed Central

    Allen, George C.; Grimm, Daniel T.; Elkan, Gerald H.

    1991-01-01

    Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 μM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 μM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 μM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min−1 mg of protein−1 and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and β-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h−1 resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C2H4h−1 mg (dry weight)−1, and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C2H4 h−1 mg (dry weight)−1. However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in “washout,” proving that ORS571 can grow on N2 and that there was not a nitrogen source in the medium that could substitute. PMID:16348585

  11. Sulphur shuttling across a chaperone during molybdenum cofactor maturation

    NASA Astrophysics Data System (ADS)

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I.; Toci, René; Mendel, Ralf R.; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

    2015-02-01

    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP—used as a surrogate of the molybdenum cofactor’s nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  12. Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

    SciTech Connect

    Sigala, Barbara; Edwards, Mina; Puri, Teena; Tsaneva, Irina R. . E-mail: tsaneva@biochem.ucl.ac.uk

    2005-11-01

    TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

  13. Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis.

    PubMed

    Probst, Corinna; Ringel, Phillip; Boysen, Verena; Wirsing, Lisette; Alexander, Mariko Matsuda; Mendel, Ralf R; Kruse, Tobias

    2014-05-01

    Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. SAGA Is a General Cofactor for RNA Polymerase II Transcription.

    PubMed

    Baptista, Tiago; Grünberg, Sebastian; Minoungou, Nadège; Koster, Maria J E; Timmers, H T Marc; Hahn, Steve; Devys, Didier; Tora, László

    2017-10-05

    Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation

    PubMed Central

    Kannemeier, Christian; Shibamiya, Aya; Nakazawa, Fumie; Trusheim, Heidi; Ruppert, Clemens; Markart, Philipp; Song, Yutong; Tzima, Eleni; Kennerknecht, Elisabeth; Niepmann, Michael; von Bruehl, Marie-Luise; Sedding, Daniel; Massberg, Steffen; Günther, Andreas; Engelmann, Bernd; Preissner, Klaus T.

    2007-01-01

    Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural “foreign surface” and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies. PMID:17405864

  16. The modulation of WTI transcription function by cofactors.

    PubMed

    Roberts, Stefan G E

    2006-01-01

    Wilms' tumour is a paediatric malignancy of the kidneys that affects one in every 10,000 live births, making it the most common solid tumour in the young. This cancer arises due to a failure of the metanephric mesenchyme to differentiate and form the kidney filtration units and tubules, which instead undergo uncontrolled proliferation. WT1 (Wilms' tumour 1) was identified as a factor that is frequently mutated in Wilms' tumours. WT1 plays a central role in the development of the genito-urinary organs and also other regions of the embryo. A major function of WT1 is to act as a regulator of transcription, controlling the expression of genes that are involved in proliferation and differentiation. WT1 can either activate or repress transcription of its target genes. Thus the transcription function of WT1 is highly context-specific, and can be modulated by a number of cofactors. Here, the known interaction partners of WT1 and the mechanisms by which they modulate WT1 transcription function will be discussed.

  17. Transcriptional and translational regulation of nitrogenase in light-dark- and continuous-light-grown cultures of the unicellular cyanobacterium Cyanothece sp. strain ATCC 51142.

    PubMed Central

    Colón-López, M S; Sherman, D M; Sherman, L A

    1997-01-01

    Cyanothece sp. strain ATCC 51142 is a unicellular, diazotrophic cyanobacterium which demonstrated extensive metabolic periodicities of photosynthesis, respiration, and nitrogen fixation when grown under N2-fixing conditions. N2 fixation and respiration peaked at 24-h intervals early in the dark or subjective-dark period, whereas photosynthesis was approximately 12 h out of phase and peaked toward the end of the light or subjective-light phase. Gene regulation studies demonstrated that nitrogenase is carefully controlled at the transcriptional and posttranslational levels. Indeed, Cyanothece sp. strain ATCC 51142 has developed an expensive mode of regulation, such that nitrogenase was synthesized and degraded each day. These patterns were seen when cells were grown under either light-dark or continuous-light conditions. Nitrogenase mRNA was synthesized from the nifHDK operon during the first 4 h of the dark period under light-dark conditions or during the first 6 h of the subjective-dark period when grown in continuous light. The nitrogenase NifH and NifDK subunits reached a maximum level at 4 to 10 h in the dark or subjective-dark periods and were shown by Western blotting and electron microscopy immunocytochemistry to be thoroughly degraded toward the end of the dark periods. An exception is the NifDK protein (MoFe-protein), which appeared not to be completely degraded under continuous-light conditions. We hypothesize that cellular O2 levels were kept low by decreasing photosynthesis and by increasing respiration in the early dark or subjective-dark periods to permit nitrogenase activity. The subsequent increase in O2 levels resulted in nitrogenase damage and eventual degradation. PMID:9209050

  18. Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

    PubMed Central

    Arias-Cartin, Rodrigo; Ceccaldi, Pierre; Schoepp-Cothenet, Barbara; Frick, Klaudia; Blanc, Jean-Michel; Guigliarelli, Bruno; Walburger, Anne; Grimaldi, Stéphane; Friedrich, Thorsten; Receveur-Brechot, Véronique; Magalon, Axel

    2016-01-01

    A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family. PMID:27886223

  19. Nanoparticle-supported multi-enzyme biocatalysis with in situ cofactor regeneration.

    PubMed

    Liu, Wenfang; Zhang, Songping; Wang, Ping

    2009-01-01

    Although there have been a long history of studying and using immobilized enzymes, little has been reported regarding the nature of immobilized cofactors. Herein we report that cofactor NAD(H) covalently attached to silica nanoparticles successfully coordinated with particle-immobilized enzymes and enabled multistep biotransformations. Specifically, silica nanoparticle-attached glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH) and NAD(H) were prepared and applied to catalyze the coupled reactions for production of alpha-ketoglutarate and lactate with the cofactor regenerated within the reaction cycle. It appeared that particle-particle collision driven by Brownian motion of the nanoparticles provided effective interactions among the catalytic components, and thus realized a dynamic shuttling of the particle-supported cofactor between the two enzymes to keep the reaction cycles continuing. Total turnover numbers (TTNs) as high as 20,000h(-1) were observed for the cofactor. It appeared to us that the use of particle-attached cofactor promises a new biochemical processing strategy for cofactor-dependent biotransformations.

  20. Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

    PubMed Central

    2011-01-01

    Background Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity. Results Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors. Conclusions Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on

  1. Cofactor Requirement of HpyAV Restriction Endonuclease

    PubMed Central

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-yong

    2010-01-01

    Background Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. Principal Findings We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Conclusions/Significance Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms. PMID:20140205

  2. HIV-1 evades innate immune recognition through specific cofactor recruitment

    NASA Astrophysics Data System (ADS)

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

    2013-11-01

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

  3. A cofactor approach to copper-dependent catalytic antibodies

    PubMed Central

    Nicholas, Kenneth M.; Wentworth, Paul; Harwig, Curtis W.; Wentworth, Anita D.; Shafton, Asher; Janda, Kim D.

    2002-01-01

    A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, LysH93, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [kcat(11) = 2.3 min−1 and Km(11) 2.2 mM] with a rate enhancement [kcat(11)kuncat(11)] of 2.1 × 105. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 × 104 in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2. PMID:11880619

  4. Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.

    PubMed

    Yang, Zhi-Yong; Ledbetter, Rhesa; Shaw, Sudipta; Pence, Natasha; Tokmina-Lukaszewska, Monika; Eilers, Brian; Guo, Qingjuan; Pokhrel, Nilisha; Cash, Valerie L; Dean, Dennis R; Antony, Edwin; Bothner, Brian; Peters, John W; Seefeldt, Lance C

    2016-07-05

    Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

  5. Characterization of Co-Cultivation of Cyanobacteria on Growth, Productions of Polysaccharides and Extracellular Proteins, Nitrogenase Activity, and Photosynthetic Activity.

    PubMed

    Xue, Chuizhao; Wang, Libo; Wu, Tong; Zhang, Shiping; Tang, Tao; Wang, Liang; Zhao, Quanyu; Sun, Yuhan

    2017-01-01

    Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N2-fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.

  6. Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase

    SciTech Connect

    Hyman, M.R.; Arp, D.J. ); Seefeldt, L.C.; Morgan, T.V.; Mortenson, L.E. )

    1992-03-24

    The effect of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C{sub 2}H{sub 2}-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactive Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicate that the effect of NO on Av2 involves oxidation of the (4Re-4S) center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. A correlation between UV-visible and EPR spectra features and the extent of NO inactivation has been established. The effects of NO on both nitrogenase components are interpreted in terms of the known reactivity of NO with Fe-S centers.

  7. Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr.

    PubMed

    Stüeken, Eva E; Buick, Roger; Guy, Bradley M; Koehler, Matthew C

    2015-04-30

    Nitrogen is an essential nutrient for all organisms that must have been available since the origin of life. Abiotic processes including hydrothermal reduction, photochemical reactions, or lightning discharge could have converted atmospheric N2 into assimilable NH4(+), HCN, or NOx species, collectively termed fixed nitrogen. But these sources may have been small on the early Earth, severely limiting the size of the primordial biosphere. The evolution of the nitrogen-fixing enzyme nitrogenase, which reduces atmospheric N2 to organic NH4(+), thus represented a major breakthrough in the radiation of life, but its timing is uncertain. Here we present nitrogen isotope ratios with a mean of 0.0 ± 1.2‰ from marine and fluvial sedimentary rocks of prehnite-pumpellyite to greenschist metamorphic grade between 3.2 and 2.75 billion years ago. These data cannot readily be explained by abiotic processes and therefore suggest biological nitrogen fixation, most probably using molybdenum-based nitrogenase as opposed to other variants that impart significant negative fractionations. Our data place a minimum age constraint of 3.2 billion years on the origin of biological nitrogen fixation and suggest that molybdenum was bioavailable in the mid-Archaean ocean long before the Great Oxidation Event.

  8. Nitrogenase activity by biological soil crusts in cold sagebrush steppe ecosystems

    USGS Publications Warehouse

    Schwabedissen, Stacy G.; Lohse, Kathleen A.; Reed, Sasha C.; Aho, Ken A.; Magnuson, Timothy S.

    2017-01-01

    In drylands worldwide, biological soil crusts (BSC) form a thin photosynthetic cover across landscapes, and provide vital benefits in terms of stabilizing soil and fixing nitrogen (N) and carbon (C). Numerous studies have examined the effects of climate and disturbance on BSC functions; however, few have characterized these responses in rolling BSCs typical of northern ecosystems in the Intermountain West, US. With temperature increases and shifts in precipitation projected, it is unclear how BSCs in this region will respond to climate change, and how the response could affect their capacity to perform key ecosystem functions, such as providing ‘new’ N through biological N2 fixation. To address this important knowledge gap, we examined nitrogenase activity (NA) associated with rolling BSCs along a climatic gradient in southwestern Idaho, US, and quantified how acetylene reduction rates changed as a function of climate, grazing (using exclosures), and shrub-canopy association. Results show that warmer, drier climates at lower elevations hosted greater cover of late successional BSC communities (e.g., mosses and lichens), and higher NA compared with colder, wetter climates at higher elevations. Highest NA (0.5–29.3 µmol C2H4 m−2 h−1) occurred during the early summer/spring, when water was more available than in late summer/autumn. Activity was strongly associated with soil characteristics including pH and ammonium concentrations suggesting these characteristics as potentially strong controls on NA in BSCs. The relationship between grazing and NA varied with elevation. Specifically, lower elevation sites had lower NA at grazed locations, whereas higher elevation sites had higher NA with grazing. At both low and high ends of the elevation gradient, shrub-canopy associated BSCs maintained two to three times higher NA compared to BSCs in the interspace among shrubs. Taken together, our findings indicate that the controls and rates of NA in BSCs vary

  9. Cofactor metals and antioxidant enzymes in cisplatin-treated rats: effect of antioxidant intervention.

    PubMed

    Sabuncuoglu, Suna; Eken, Ayse; Aydin, Ahmet; Ozgunes, Hilal; Orhan, Hilmi

    2015-10-01

    We explored the association between the activities of antioxidant enzymes and their metallic cofactors in rats treated with cisplatin. The antioxidant effects of aminoguanidine, and a combination of vitamins E and C were investigated. Plasma platin was significantly lower than liver and kidney. Cisplatin treatment caused significant increase in plasma Se-glutathione peroxidase activity. Activities of Se-glutathione peroxidase, glutathione S-transferase, catalase and Cu,Zn-superoxide dismutase have been found to be significantly decreased in liver and kidney compared to controls. Zn levels in these organs were diminished upon cisplatin treatment, while levels of Cu were unaffected. Interestingly, levels of iron, the cofactor of catalase, were found to be significantly increased in liver and kidney. Intervention with aminoguanidine or vitamins was generally prevented cisplatin-caused changes in the activity of enzymes and in the tissue levels of cofactor metals. These observations suggest that relation between activities of enzymes and levels of cofactor metals is multifactorial.

  10. The effect of ribosome assembly cofactors on in vitro 30S subunit reconstitution.

    PubMed

    Bunner, Anne E; Nord, Stefan; Wikström, P Mikael; Williamson, James R

    2010-04-23

    Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimP enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimM caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis. (c) 2010 Elsevier Ltd. All rights reserved.

  11. Understanding Dermatan Sulfate-Heparin Cofactor II Interaction through Virtual Library Screening.

    PubMed

    Raghuraman, Arjun; Mosier, Philip D; Desai, Umesh R

    2010-09-09

    Dermatan sulfate, an important member of the glycosaminoglycan family, interacts with heparin cofactor II, a member of the serpin family of proteins, to modulate antithrombotic response. Yet, the nature of this interaction remains poorly understood at a molecular level. We report the genetic algorithm-based combinatorial virtual library screening study of a natural, high-affinity dermatan sulfate hexasaccharide with heparin cofactor II. Of the 192 topologies possible for the hexasaccharide, only 16 satisfied the "high-specificity" criteria used in computational study. Of these, 13 topologies were predicted to bind in the heparin-binding site of heparin cofactor II at a ∼60° angle to helix D, a novel binding mode. This new binding geometry satisfies all known solution and mutagenesis data and supports thrombin ternary complexation through a template mechanism. The study is expected to facilitate the design of allosteric agonists of heparin cofactor II as antithrombotic agents.

  12. Interactions of the Mcm1 MADS Box Protein with Cofactors That Regulate Mating in Yeast

    PubMed Central

    Mead, Janet; Bruning, Adrian R.; Gill, Michael K.; Steiner, Andrew M.; Acton, Thomas B.; Vershon, Andrew K.

    2002-01-01

    The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the α1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the α2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact α2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with α2 than for interaction with either α1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors. PMID:12052870

  13. Reciprocal light-dark transcriptional control of nif and rbc expression and light-dependent posttranslational control of nitrogenase activity in Synechococcus sp. strain RF-1.

    PubMed

    Chow, T J; Tabita, F R

    1994-10-01

    Synechococcus sp. strain RF-1 exhibits a circadian rhythm of N2 fixation when cells are grown under a light-dark cycle, with nitrogenase activity observed only during the dark period. This dark-dependent activity correlated with nif gene transcription in strain RF-1. By using antibodies against dinitrogenase reductase (the Fe protein of the nitrogenase complex), it was found that there was a distinct shift in the mobility of this protein on sodium dodecyl sulfate gels during the light-dark cycle. The Fe protein was present only when cells were incubated in the dark. Upon illumination, there was a conversion of all Fe protein to a modified form, after which it rapidly disappeared from extracts. These studies indicated that all nitrogenase activity present during the dark cycle resulted from de novo synthesis of nitrogenase. Upon entering the light phase, cells appeared to quickly degrade the modified form of Fe protein, perhaps as a result of activating or inducing a protease. By contrast, transcription of the rbcL gene, which encodes the catalytic subunit of the key enzyme of CO2 fixation (a light-dependent process), was enhanced in the light.

  14. The glmS Ribozyme Cofactor is a General Acid-Base Catalyst

    PubMed Central

    Viladoms, Julia; Fedor, Martha J.

    2012-01-01

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

  15. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme

    PubMed Central

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-01-01

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg2+ ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg2+ ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process. PMID:27440438

  16. A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

    PubMed

    Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

    2017-02-17

    The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

  17. The glmS ribozyme cofactor is a general acid-base catalyst.

    PubMed

    Viladoms, Júlia; Fedor, Martha J

    2012-11-21

    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

  18. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex

    PubMed Central

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  19. Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol.

    PubMed

    Wang, Pengchao; Yang, Xinwei; Lin, Baixue; Huang, Jianzhong; Tao, Yong

    2017-09-22

    The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A "bridge" between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals. Copyright © 2017. Published by Elsevier Inc.

  20. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    PubMed

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009.

    PubMed

    Calusinska, Magdalena; Hamilton, Christopher; Monsieurs, Pieter; Mathy, Gregory; Leys, Natalie; Franck, Fabrice; Joris, Bernard; Thonart, Philippe; Hiligsmann, Serge; Wilmotte, Annick

    2015-01-01

    Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. This work, contributing to

  2. Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase.

    PubMed

    Maritano, S; Fairhurst, S A; Eady, R R

    2001-06-01

    We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the [4Fe-4S]+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1). Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times. These changes were not associated with catalysis since no evolution of H2 was detectable.

  3. Mössbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions

    PubMed Central

    Smith, Barry E.; Lang, George

    1974-01-01

    The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in 57Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na2S2O4, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe4S4 or Fe2S2 low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth's Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na2S2O4, ATP and Mg2+, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na2S2O4-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all

  4. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase

    SciTech Connect

    Warren, M.J.; Jordan, P.M.

    1988-12-13

    The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA/sup -/ mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free ..cap alpha..-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino(5-/sup 14/C)levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the ..cap alpha..-substituted substrate analogue ..cap alpha..-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cummulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH/sub 3/ or H/sub 2/O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

  5. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    PubMed

    Ghosh, Amit; Zhao, Huimin; Price, Nathan D

    2011-01-01

    Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  6. Genome-Scale Consequences of Cofactor Balancing in Engineered Pentose Utilization Pathways in Saccharomyces cerevisiae

    PubMed Central

    Ghosh, Amit; Zhao, Huimin; Price, Nathan D.

    2011-01-01

    Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment. PMID:22076150

  7. Elucidating thermodynamic parameters for electron transfer proteins using isothermal titration calorimetry: application to the nitrogenase Fe protein.

    PubMed

    Sørlie, Morten; Chan, Jeannine M; Wang, Haijang; Seefeldt, Lance C; Parker, Vernon D

    2003-05-01

    Establishing thermodynamic parameters for electron transfer reactions involving redox proteins is essential for a complete description of these important reactions. While various methods have been developed for measuring the Gibbs free energy change (Delta G(HR) or E(m)) for the protein half-reactions, deconvolution of the respective contributions of enthalpy (Delta H(HR)) and entropy (Delta S(HR)) changes is much more challenging. In the present work, an approach is developed using isothermal titration calorimetry (ITC) that allows accurate determination of all of these thermodynamic parameters for protein electron transfer half-reactions. The approach was validated for essentially irreversible and reversible electron transfer reactions between well-characterized mediators and between mediators and the protein cytochrome c. In all cases, the measured thermodynamic parameters were in excellent agreement with parameters determined by electrochemical methods. Finally, the calorimetry approach was used to determine thermodynamic parameters for electron transfer reactions of the nitrogenase Fe protein [4Fe-4S](2+/+) couple in the absence or presence of MgADP or MgATP. The E(m) value was found to change from -290 mV in the absence of nucleotides to -381 mV with MgATP and -423 mV with MgADP, consistent with earlier values. For the first time, the enthalpy (Delta H(HR)) and entropy (Delta S(HR)) contributions for each case were established, revealing shifts in the contribution of each thermodynamic parameter induced by nucleotide binding. The results are discussed in the context of current models for electron transfer in nitrogenase.

  8. A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

    PubMed Central

    Zhao, Dehua; Liu, Xiaomeng; Zhang, Bo; Xie, Jianbo; Hong, Yuanyuan; Li, Pengfei; Chen, Sanfeng; Dixon, Ray; Li, Jilun

    2013-01-01

    Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes. PMID:24146630

  9. Incorporation of different N sources and light response curves of nitrogenase and photosynthesis by cyanobacterial blooms from rice fields.

    PubMed

    Ariosa, Yoanna; Carrasco, David; Quesada, Antonio; Fernández-Valiente, Eduardo

    2006-04-01

    In this work, we estimate the contributions of the different sources of N incorporated by two N(2)-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N(2)-fixing cyanobacterial blooms in rice fields comes from N(2) fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N(2) fixation (only about 33-41% of the N incorporated came from N(2) fixation). Our results on the photodependence of C and N(2) fixation indicate that in both cyanobacterial blooms, N(2) fixation showed a steeper initial slope (alpha) and was saturated with less irradiance than C fixation, suggesting that N(2) fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N(2) fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.

  10. A new cofactor in prokaryotic enzyme: Tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase

    SciTech Connect

    McIntire, W.S. Univ. of California, San Francisco ); Wemmer, D.E. ); Chistoserdov, A.; Lidstrom, M.E. )

    1991-05-10

    Methylamine dehydrogenase (MADH), an {alpha}{sub 2}{beta}{sub 2} enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small {beta} subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting {sup 1}H and {sup 13}C nuclear magnetic resonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, of pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.

  11. Regulation of Estrogen-Dependent Transcription by the LIM Cofactors CLIM and RLIM in Breast Cancer

    PubMed Central

    Johnsen, Steven A.; Güngör, Cenap; Prenzel, Tanja; Riethdorf, Sabine; Riethdorf, Lutz; Taniguchi-Ishigaki, Naoko; Rau, Thomas; Tursun, Baris; Furlow, J. David; Sauter, Guido; Scheffner, Martin; Pantel, Klaus; Gannon, Frank; Bach, Ingolf

    2009-01-01

    Mammary oncogenesis is profoundly influenced by signaling pathways controlled by Estrogen Receptor-alpha (ERα). Although it is known that ERα exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HDs) is critically regulated by Cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERα cofactors that co-localize and interact with ERα in primary human breast tumors. We show that both cofactors associate with estrogen responsive promoters and regulate the expression of endogenous ERα target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERα and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERα on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs, but enhances transcriptional activation of endogenous ERα target genes. Results from a human breast cancer tissue microarray (TMA) of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/PR positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERα during the development of human breast cancer. PMID:19117995

  12. Cofactor-specific photochemical function resolved by ultrafast spectroscopy in photosynthetic reaction center crystals.

    PubMed

    Huang, Libai; Ponomarenko, Nina; Wiederrecht, Gary P; Tiede, David M

    2012-03-27

    High-resolution mapping of cofactor-specific photochemistry in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was achieved by polarization selective ultrafast spectroscopy in single crystals at cryogenic temperature. By exploiting the fixed orientation of cofactors within crystals, we isolated a single transition within the multicofactor manifold, and elucidated the site-specific photochemical functions of the cofactors associated with the symmetry-related active A and inactive B branches. Transient spectra associated with the initial excited states were found to involve a set of cofactors that differ depending upon whether the monomeric bacteriochlorophylls, BChl(A), BChl(B), or the special pair bacteriochlorophyll dimer, P, was chosen for excitation. Proceeding from these initial excited states, characteristic photochemical functions were resolved. Specifically, our measurements provide direct evidence for an alternative charge separation pathway initiated by excitation of BChl(A) that does not involve P*. Conversely, the initial excited state produced by excitation of BChl(B) was found to decay by energy transfer to P. A clear sequential kinetic resolution of BChl(A) and the A-side bacteriopheophytin, BPh(A), in the electron transfer proceeding from P* was achieved. These experiments demonstrate the opportunity to resolve photochemical function of individual cofactors within the multicofactor RC complexes using single crystal spectroscopy.

  13. Cofactor dependence and isotype distribution of anticardiolipin antibodies in viral infections

    PubMed Central

    Guglielmone, H; Vitozzi, S; Elbarcha, O; Fernandez, E

    2001-01-01

    BACKGROUND—Antibodies to cardiolipin (aCLs) are often detected in patients with autoimmune disorders or infectious diseases.
OBJECTIVE—To investigate the distribution of aCL isotypes and requirement of protein cofactor in viral infections in order to establish the importance, if any, of these antibodies in these infectious diseases.
PATIENTS AND METHODS—The isotype distribution of aCLs in the sera from 160 patients with infection caused by HIV-1 (n=40), hepatitis A virus (n=40), hepatitis B virus (n=40), or hepatitis C virus (n=40) was studied by standardised enzyme linked immunosorbent assay (ELISA) in the presence and absence of protein cofactor (mainly β2-glycoprotein I). Serum samples from healthy volunteers and patients with syphilis and antiphospholipid syndrome were also included and served as negative and positive control groups respectively.
RESULTS—The prevalence of one or more aCL isotypes in serum of patients with HIV-1, hepatitis A virus, hepatitis B virus, or hepatitis C virus infection was 47%, 92%, 42%, and 17% respectively (principally IgM and/or IgA). Most of these antibodies were mainly cofactor independent.
CONCLUSIONS—The presence of aCLs in viral infections is principally cofactor independent, suggesting that cofactor dependence of the aCLs should be assessed to distinguish subjects most likely to suffer from clinical symptoms observed in the presence of these antibodies.

 PMID:11302873

  14. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

  15. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    PubMed

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Cofactor regeneration in phototrophic cyanobacteria applied for asymmetric reduction of ketones.

    PubMed

    Havel, Jan; Weuster-Botz, Dirk

    2007-07-01

    The obligate photoautotrophic cyanobacterium Synechococcus PCC7942 and the photoheterotrophic heterocystous cyanobacterium Noctoc muscorum are able to reduce prochiral ketones asymmetrically to optical pure chiral alcohols without light. An example is the synthesis of S-pentafluoro(phenyl-)ethanol with an enantiomeric excess >99% if 2'-3'-4'-5'-6'-pentafluoroacetophenone is used as substrate. If no light is available for regeneration of the cofactor nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), glucose is used as cosubstrate. Membrane disintegration during asymmetric reduction promotes cytosolic energy generating metabolic pathways. Observed regulatory effects depicted by an adenosine triphosphate (ATP) to nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP(+)) ratio of 3:1 for efficient cofactor recycling indicate a metabolization via glycolisis. The stoichiometric formation of the by-product acetate (1 mol acetate/1 mol chiral alcohol) indicates homoacetic acid fermentation for cofactor regeneration including the obligate photoautotrophic cyanobacterium Synechococcus PCC7942.

  17. Anthocyanin copigmentation and color of wine: The effect of naturally obtained hydroxycinnamic acids as cofactors.

    PubMed

    Bimpilas, Andreas; Panagopoulou, Marilena; Tsimogiannis, Dimitrios; Oreopoulou, Vassiliki

    2016-04-15

    Copigmentation of anthocyanins accounts for over 30% of fresh red wine color, while during storage, the color of polymeric pigments formed between anthocyanins and proanthocyanidins predominates. Rosmarinic acid and natural extracts rich in hydroxycinnamic acids, obtained from aromatic plants (Origanum vulgare and Satureja thymbra), were examined as cofactors to fresh Merlot wine and the effect on anthocyanin copigmentation and wine color was studied during storage for 6months. An increase of the copigmented anthocyanins that enhanced color intensity by 15-50% was observed, confirming the ability of complex hydroxycinnamates to form copigments. The samples with added cofactors retained higher percentages of copigmented anthocyanins and higher color intensity, compared to the control wine, up to 3 months. However, the change in the equilibrium between monomeric and copigmented anthocyanins that was induced by added cofactors, did not affect the rate of polymerization reactions during storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The Tight Junction Proteins Claudin-1, -6, and -9 Are Entry Cofactors for Hepatitis C Virus▿

    PubMed Central

    Meertens, Laurent; Bertaux, Claire; Cukierman, Lisa; Cormier, Emmanuel; Lavillette, Dimitri; Cosset, François-Loïc; Dragic, Tatjana

    2008-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81+ cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis. PMID:18234789

  19. Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

    PubMed Central

    Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

    2016-01-01

    In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

  20. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

    PubMed

    Prier, Christopher K; Arnold, Frances H

    2015-11-11

    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  1. The phylogenomic roots of modern biochemistry: origins of proteins, cofactors and protein biosynthesis.

    PubMed

    Caetano-Anollés, Gustavo; Kim, Kyung Mo; Caetano-Anollés, Derek

    2012-02-01

    The complexity of modern biochemistry developed gradually on early Earth as new molecules and structures populated the emerging cellular systems. Here, we generate a historical account of the gradual discovery of primordial proteins, cofactors, and molecular functions using phylogenomic information in the sequence of 420 genomes. We focus on structural and functional annotations of the 54 most ancient protein domains. We show how primordial functions are linked to folded structures and how their interaction with cofactors expanded the functional repertoire. We also reveal protocell membranes played a crucial role in early protein evolution and show translation started with RNA and thioester cofactor-mediated aminoacylation. Our findings allow elaboration of an evolutionary model of early biochemistry that is firmly grounded in phylogenomic information and biochemical, biophysical, and structural knowledge. The model describes how primordial α-helical bundles stabilized membranes, how these were decorated by layered arrangements of β-sheets and α-helices, and how these arrangements became globular. Ancient forms of aminoacyl-tRNA synthetase (aaRS) catalytic domains and ancient non-ribosomal protein synthetase (NRPS) modules gave rise to primordial protein synthesis and the ability to generate a code for specificity in their active sites. These structures diversified producing cofactor-binding molecular switches and barrel structures. Accretion of domains and molecules gave rise to modern aaRSs, NRPS, and ribosomal ensembles, first organized around novel emerging cofactors (tRNA and carrier proteins) and then more complex cofactor structures (rRNA). The model explains how the generation of protein structures acted as scaffold for nucleic acids and resulted in crystallization of modern translation.

  2. ModE-dependent molybdate regulation of the molybdenum cofactor operon moa in Escherichia coli.

    PubMed

    Anderson, L A; McNairn, E; Lubke, T; Pau, R N; Boxer, D H; Leubke, T

    2000-12-01

    The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the -35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moa expression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.

  3. Identification of a Bis-molybdopterin Intermediate in Molybdenum Cofactor Biosynthesis in Escherichia coli*

    PubMed Central

    Reschke, Stefan; Sigfridsson, Kajsa G. V.; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke

    2013-01-01

    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo–S and Mo–O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. PMID:24003231

  4. ModE-Dependent Molybdate Regulation of the Molybdenum Cofactor Operon moa in Escherichia coli

    PubMed Central

    Anderson, Lisa A.; McNairn, Elizabeth; Leubke, Torben; Pau, Richard N.; Boxer, David H.

    2000-01-01

    The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moa expression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression of moa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium. PMID:11092866

  5. Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.

    PubMed

    Reschke, Stefan; Sigfridsson, Kajsa G V; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke

    2013-10-11

    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

  6. Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor.

    PubMed

    Griese, Julia J; Kositzki, Ramona; Schrapers, Peer; Branca, Rui M M; Nordström, Anders; Lehtiö, Janne; Haumann, Michael; Högbom, Martin

    2015-10-16

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn(II) and Fe(II) in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189-17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor*

    PubMed Central

    Griese, Julia J.; Kositzki, Ramona; Schrapers, Peer; Branca, Rui M. M.; Nordström, Anders; Lehtiö, Janne; Haumann, Michael; Högbom, Martin

    2015-01-01

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from MnII and FeII in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R. M., Lehtiö, J., Gräslund, A., Lubitz, W., Siegbahn, P. E., and Högbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 17189–17194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability. PMID:26324712

  8. Redirecting metabolic flux in Saccharomyces cerevisiae through regulation of cofactors in UMP production.

    PubMed

    Chen, Yong; Liu, Qingguo; Chen, Xiaochun; Wu, Jinglan; Guo, Ting; Zhu, Chenjie; Ying, Hanjie

    2015-04-01

    Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. To understand the impact of cofactors on physiological functions, a systematic approach was applied, which involved redox state analysis, energy charge analysis, and metabolite analysis. Using uridine 5'-monophosphate metabolism in Saccharomyces cerevisiae as a model, we demonstrated that regulation of intracellular the ratio of NADPH to NADP(+) not only redistributed the carbon flux between the glycolytic and pentose phosphate pathways, but also regulated the redox state of NAD(H), resulting in a significant change of ATP, and a significantly altered spectrum of metabolic products.

  9. A global transcription cofactor bound to juxtaposed strands of unwound DNA.

    PubMed

    Werten, Sebastiaan; Moras, Dino

    2006-02-01

    The 1.74-A crystal structure of the human transcription cofactor PC4 in complex with a single-stranded 20-mer oligonucleotide reveals how symmetry-related beta-surfaces of the protein homodimer interact with juxtaposed 5-nucleotide DNA regions running in opposite directions. The structure explains high-affinity binding of PC4 to the complementary strands of unwinding duplex DNA, and it suggests the cofactor may have a role in relaxing negative supercoils or exposing unpaired bases for sequence-specific recognition by other biomolecules.

  10. Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

    PubMed

    Marsden, B J; Sauer, F D; Blackwell, B A; Kramer, J K

    1989-03-31

    The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

  11. Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors

    PubMed Central

    Zinder, John C.

    2017-01-01

    The eukaryotic RNA exosome is an essential and conserved protein complex that can degrade or process RNA substrates in the 3′-to-5′ direction. Since its discovery nearly two decades ago, studies have focused on determining how the exosome, along with associated cofactors, achieves the demanding task of targeting particular RNAs for degradation and/or processing in both the nucleus and cytoplasm. In this review, we highlight recent advances that have illuminated roles for the RNA exosome and its cofactors in specific biological pathways, alongside studies that attempted to dissect these activities through structural and biochemical characterization of nuclear and cytoplasmic RNA exosome complexes. PMID:28202538

  12. NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii.

    PubMed

    Johnson, D C; Dos Santos, P C; Dean, D R

    2005-02-01

    In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.

  13. Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium.

    PubMed

    Loiret, F G; Grimm, B; Hajirezaei, M R; Kleiner, D; Ortega, E

    2009-07-15

    Pantoea sp. is an endophytic nitrogen-fixing bacterium isolated from sugarcane tissues. The aim of the present study was to determine the contents of amino acids in sugarcane as a result of inoculation of nodes and nodal roots with Pantoea sp. strain 9C and to evaluate the effects of amino acids on growth, nitrogenase activity and ammonium excretion of the bacterium. Content of almost all amino acids increased in 30-day-old plantlets by root inoculation. The most abundant amino acids in shoot tissues were asparagine and proline, and those in nodal roots were asparagine, proline, aspartic acid, glutamic acid and serine. The bacterium was able to grow on all tested amino acids except histidine, isoleucine and leucine. Nitrogenase Pantoea sp. was partially inhibited by 1, 2 or 5mmolL(-1) and completely inhibited by 10mmolL(-1) of NH(4)(+) in the media. Pantoea sp. showed nitrogenase activity in 5mmolL(-1) of serine, asparagine, threonine, alanine, proline, tyrosine, valine, methionine, lysine, phenylalanine, cysteine, tryptophan, citrulline and ornithine. Pantoea sp. did not excrete ammonium when it grew in vivo conditions favoring nitrogen fixation; however, ammonium was detected in the supernatant when 5mmolL(-1) asparagine, aspartic acid, alanine, serine or glutamine was added to the medium. The highest ammonium concentration in the supernatant was detected, when Pantoea grew on serine. Ammonium in the supernatant and nitrogenase activity were only detectable concomitantly when the medium was supplemented with serine, alanine, glutamine or asparagine. We discuss roles of amino acids on plant-bacteria interaction during the colonization of sugarcane plants.

  14. Uncoupling nitrogenase: catalytic reduction of hydrazine to ammonia by a MoFe protein in the absence of Fe protein-ATP.

    PubMed

    Danyal, Karamatullah; Inglet, Boyd S; Vincent, Kylie A; Barney, Brett M; Hoffman, Brian M; Armstrong, Fraser A; Dean, Dennis R; Seefeldt, Lance C

    2010-09-29

    The catalytic reduction of hydrazine (N(2)H(4)) to ammonia by a β-98(Tyr→His) MoFe protein in the absence of the Fe protein or ATP is reported. The reduction of N(2) or other substrates (e.g., hydrazine, protons, acetylene) by nitrogenase normally requires the transient association of the two nitrogenase component proteins, the Fe protein and the MoFe protein. The Fe protein, with two bound MgATP molecules, transfers one electron to the MoFe protein during each association, coupled to the hydrolysis of two MgATP. All substrate reduction reactions catalyzed by nitrogenase require delivery of electrons by the Fe protein coupled to the hydrolysis of MgATP. We report that when a single amino acid within the MoFe protein (β-98(Tyr)) is substituted by His, the resulting MoFe protein supports catalytic reduction of the nitrogenous substrate hydrazine (N(2)H(4)) to two ammonia molecules when provided with a low potential reductant, polyaminocarboxylate ligated Eu(II) (E(m) -1.1 V vs NHE). The wild-type and a number of other MoFe proteins with amino acid substitutions do not show significant rates of hydrazine reduction under these conditions, whereas the β-98(His) MoFe protein catalyzes hydrazine reduction at rates up to 170 nmol NH(3)/min/mg MoFe protein. This rate of hydrazine reduction is 94% of the rate catalyzed by the β-98(His) or wild-type MoFe protein when combined with the Fe protein, ATP, and reductant under comparable conditions. The β-98(His) MoFe protein reduction of hydrazine in the absence of the Fe protein showed saturation kinetics for the concentration of reductant and substrate. The implications of these results in understanding the nitrogenase mechanism are discussed.

  15. Endomicrobium proavitum, the first isolate of Endomicrobia class. nov. (phylum Elusimicrobia)--an ultramicrobacterium with an unusual cell cycle that fixes nitrogen with a Group IV nitrogenase.

    PubMed

    Zheng, Hao; Dietrich, Carsten; Radek, Renate; Brune, Andreas

    2016-01-01

    The bacterial tree contains many deep-rooting clades without any cultured representatives. One such clade is 'Endomicrobia', a class-level lineage in the phylum Elusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free-living member of this clade from sterile-filtered gut homogenate of defaunated (starch-fed) Reticulitermes santonensis. Strain Rsa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO2. Ultrastructural analysis revealed a Gram-negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of Group IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and (15)N2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to 'Candidatus Endomicrobium trichonymphae' and other obligate endosymbionts, we propose the name 'Endomicrobium proavitum' gen. nov., sp. nov. for the first isolate of this lineage and the name 'Endomicrobia' class. nov. for the entire clade.

  16. A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates.

    PubMed

    Lukoyanov, Dmitriy; Yang, Zhi-Yong; Duval, Simon; Danyal, Karamatullah; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

    2014-04-07

    We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1-8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped "hydride" state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.

  17. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition.

    PubMed

    Payne, Karl A P; White, Mark D; Fisher, Karl; Khara, Basile; Bailey, Samuel S; Parker, David; Rattray, Nicholas J W; Trivedi, Drupad K; Goodacre, Royston; Beveridge, Rebecca; Barran, Perdita; Rigby, Stephen E J; Scrutton, Nigel S; Hay, Sam; Leys, David

    2015-06-25

    The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

  18. Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

    PubMed Central

    Haft, Daniel H.; Pierce, Phillip G.; Mayclin, Stephen J.; Sullivan, Amy; Gardberg, Anna S.; Abendroth, Jan; Begley, Darren W.; Phan, Isabelle Q.; Staker, Bart L.; Myler, Peter J.; Marathias, Vasilios M.; Lorimer, Donald D.; Edwards, Thomas E.

    2017-01-01

    During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin. PMID:28120876

  19. Artificial photosynthesis on a chip: microfluidic cofactor regeneration and photoenzymatic synthesis under visible light.

    PubMed

    Lee, Joon Seok; Lee, Sahng Ha; Kim, Jae Hong; Park, Chan Beum

    2011-07-21

    We present a microfluidic artificial photosynthetic platform that incorporates quantum dots and redox enzymes for photoenzymatic synthesis of fine chemicals under visible light. Similar to natural photosynthesis, photochemical cofactor regeneration takes place in the light-dependent reaction zone, which is then coupled with the light-independent, enzymatic synthesis in the downstream of the microchannel.

  20. Engineering the Assembly of Heme Cofactors in Man-Made Proteins

    PubMed Central

    2015-01-01

    Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways. PMID:24495285

  1. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

    PubMed Central

    Payne, Karl A.P.; White, Mark D.; Fisher, Karl; Khara, Basile; Bailey, Samuel S.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Beveridge, Rebecca; Barran, Perdita; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

    2016-01-01

    The ubiD/ubiX or the homologous fdc/pad genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis1–3 or microbial biodegradation of aromatic compounds4–6 respectively. Despite biochemical studies on individual gene products, the composition and co-factor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7–9. We show Fdc is solely responsible for (de)carboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesised by the associated UbiX/Pad10. Atomic resolution crystal structures reveal two distinct isomers of the oxidized cofactor can be observed: an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with drastically altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. While 1,3-dipolar cycloaddition is commonly used in organic chemistry11–12, we propose this presents the first example of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation. PMID:26083754

  2. Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

    PubMed

    Patton, Gregory C; Stenmark, Pål; Gollapalli, Deviprasad R; Sevastik, Robin; Kursula, Petri; Flodin, Susanne; Schuler, Herwig; Swales, Colin T; Eklund, Hans; Himo, Fahmi; Nordlund, Pär; Hedstrom, Lizbeth

    2011-10-30

    Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

  3. Tetrahydropterin as a possible natural cofactor in the drosophila phenylalanine hydroxylation system

    SciTech Connect

    Bel, Y.; Jacobson, K.B.; Ferre, J. . Dept. of Genetics; Oak Ridge National Lab., TN; Valencia Univ. . Dept. of Genetics)

    1989-01-01

    The aim of the present work is the study of phenylalanine hydroxylase (PH) activity of Drosophila melanogaster wild type with different cofactors: the two natural occurring tetrahydropteridines (BH{sub 4} and PH{sub 4}) and the synthetic 6,7-dimethyltetrahydropterin (DMPH{sub 4}), as well as the determination of this activity at different developmental stages. 7 refs., 2 figs.

  4. Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways

    PubMed Central

    Heininger, Annika U.; Hackert, Philipp; Andreou, Alexandra Z.; Boon, Kum-Loong; Memet, Indira; Prior, Mira; Clancy, Anne; Schmidt, Bernhard; Urlaub, Henning; Schleiff, Enrico; Sloan, Katherine E.; Deckers, Markus; Lührmann, Reinhard; Enderlein, Jörg; Klostermeier, Dagmar; Rehling, Peter; Bohnsack, Markus T.

    2016-01-01

    ABSTRACT A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes. PMID:26821976

  5. The archaeal cofactor F0 is a light-harvesting antenna chromophore in eukaryotes.

    PubMed

    Glas, Andreas F; Maul, Melanie J; Cryle, Max; Barends, Thomas R M; Schneider, Sabine; Kaya, Emine; Schlichting, Ilme; Carell, Thomas

    2009-07-14

    Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F(0)) and F(420) are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F(420)/F(420)H(2). In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F(0). Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F(0) in complex with the D. melanogaster protein shows the atomic details of F(0) binding and activation, allowing characterization of the residues involved in F(0) activation. The results show that the F(0)/F(420) coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F(0) cofactor also in higher eukaryotes.

  6. Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity*

    PubMed Central

    Lecerf, Maxime; Scheel, Tobias; Pashov, Anastas D.; Jarossay, Annaelle; Ohayon, Delphine; Planchais, Cyril; Mesnage, Stephane; Berek, Claudia; Kaveri, Srinivas V.; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2015-01-01

    The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses. PMID:25564611

  7. A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination.

    PubMed

    Gand, Martin; Thöle, Christian; Müller, Hubertus; Brundiek, Henrike; Bashiri, Ghader; Höhne, Matthias

    2016-07-20

    Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.

  8. Engineering the assembly of heme cofactors in man-made proteins.

    PubMed

    Solomon, Lee A; Kodali, Goutham; Moser, Christopher C; Dutton, P Leslie

    2014-02-26

    Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways.

  9. Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

    PubMed

    Bae, Sang-Woo; Eom, Doyoung; Mai, Ngoc Lan; Koo, Yoon-Mo

    2016-03-01

    The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.

  10. Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

    PubMed Central

    Sanchez, Isabelle; Dequin, Sylvie; Camarasa, Carole

    2015-01-01

    Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD+/NADH or, to a lesser extent, NADP+/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation. PMID:26475113

  11. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    PubMed

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

    PubMed Central

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha

    2012-01-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)+ to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+ (S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, where Km,NAD is the Km for NAD+ and Km,NADP is the Km for NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+ as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156. PMID:22344653

  13. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.

    PubMed

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung-Kul

    2012-05-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

  14. The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.

    PubMed

    Ney, Blair; Ahmed, F Hafna; Carere, Carlo R; Biswas, Ambarish; Warden, Andrew C; Morales, Sergio E; Pandey, Gunjan; Watt, Stephen J; Oakeshott, John G; Taylor, Matthew C; Stott, Matthew B; Jackson, Colin J; Greening, Chris

    2017-01-01

    F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

  15. Cell-free activation of phagocyte NADPH-oxidase: tissue and differentiation-specific expression of cytosolic cofactor activity.

    PubMed

    Parkinson, J F; Akard, L P; Schell, M J; Gabig, T G

    1987-06-30

    We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.

  16. N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

    USGS Publications Warehouse

    Bazylinski, D.A.; Dean, A.J.; Schuler, D.; Phillips, E.J.P.; Lovley, D.R.

    2000-01-01

    Cells of Geobacter metallireducens, Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N2-dependent growth, the first anaerobically with Fe(lll) as the electron acceptor, and the latter three species micro-aerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N2 under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ?? 5.9nmol C2H4 produced min-1 mg-1 cell protein) while growing with Fe(lll) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum, indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum, fix atmospheric dinitrogen.

  17. Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry 1

    PubMed Central

    Tjepkema, John D.; Cartica, Robert J.

    1982-01-01

    Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O2 partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O2 microelectrode measurements which showed that the O2 partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O2 uptake as a function of temperature and partial pressure of O2 were consistent with diffusion limitation of O2 uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O2 regulation in legume and Parasponia nodules is very similar and differs from O2 regulation in actionorhizal nodules. Images PMID:16662284

  18. Estimation of nitrogenase activity in the presence of ethylene biosynthesis by use of deuterated acetylene as a substrate

    SciTech Connect

    Lin-Vien, D.; Fateley, W.G.; Davis, L.C. )

    1989-02-01

    Nitrogenase reduces deuterated acetylene primarily to cis dideuterated ethylene. This can be distinguished from undeuterated ethylene by the use of Fourier transform infrared spectroscopy. Characteristic bands in the region from 800 to 3,500 cm-1 can be used to identify and quantitate levels of these products. This technique is applicable to field studies of nitrogen fixation where ethylene biosynthesis by plants or bacteria is occurring. We have verified the reaction stoichiometry by using Klebsiella pneumoniae and Bradyrhizobium japonicum in soybeans. The most useful bands for quantitation of substrate purity and product distribution are as follows: acetylene-d0, 3,374 cm-1; acetylene-d1, 2,584 cm-1; acetylene-d2, 2,439 cm-1; cis-ethylene-d2, 843 cm-1; trans-ethylene-d2, 988 cm-1; ethylene-d1, 943 cm-1; ethylene-d0, 949 cm-1. (The various deuterated ethylenes and acetylenes are designated by a lowercase d and subscript to indicate the number, but not the position, of deuterium atoms in the molecule.) Mass spectrometry coupled to a gas chromatograph system has been used to assist in quantitation of the substrate and product distributions. Significant amounts of trans-ethylene-d2 were produced by both wild-type and nifV mutant K. pneumoniae. Less of this product was observed with the soybean system.

  19. Interactions between Lugol's fixative and ethylene in the acetylene-reduction assay for nitrogenase activity in lake water.

    PubMed

    Leonardson, L

    1980-05-01

    Lugol's solution is a practical and efficient fixative for the acetylene-reduction assay of nitrogenase activity in aquatic organisms. Correction must be made, however, for the solubility of ethylene in the liquid phase and reactions between Lugol's solution and ethylene. With a vapor phase-liquid phase volume ratio of 1.9:1, the mean solubility of ethylene in mixtures of lake water and Lugol's solution was 7.2%. No correlation was found between ethylene solubility and the concentration of Lugol's solution. Storage of fixed samples for more than 1 day before gas chromatographic analysis resulted in increased loss of ethylene from the vapor phase; the loss amounted to ca. 18% after 3 days. Higher losses were noted at higher concentrations of Lugol's solution. Most probably these effects were caused by iodine addition to ethylene, as indicated by the consumption of ethylene by iodine-potassium iodide solutions. The reaction was catalyzed by the rubber septa of the incubaton vessels when the septa were in contact with the liquid phase. Loss of ethylene decreased with increased concentration of phytoplankton because the organisms absorbed iodine. By using a standardized technique and determining ethylene solubility and reaction patterns between ethylene and the mixture of water and Lugol's solution, it is possible to correct for the loss of ethylene.

  20. Estimation of nitrogenase activity in the presence of ethylene biosynthesis by use of deuterated acetylene as a substrate.

    PubMed Central

    Lin-Vien, D; Fateley, W G; Davis, L C

    1989-01-01

    Nitrogenase reduces deuterated acetylene primarily to cis dideuterated ethylene. This can be distinguished from undeuterated ethylene by the use of Fourier transform infrared spectroscopy. Characteristic bands in the region from 800 to 3,500 cm-1 can be used to identify and quantitate levels of these products. This technique is applicable to field studies of nitrogen fixation where ethylene biosynthesis by plants or bacteria is occurring. We have verified the reaction stoichiometry by using Klebsiella pneumoniae and Bradyrhizobium japonicum in soybeans. The most useful bands for quantitation of substrate purity and product distribution are as follows: acetylene-d0, 3,374 cm-1; acetylene-d1, 2,584 cm-1; acetylene-d2, 2,439 cm-1; cis-ethylene-d2, 843 cm-1; trans-ethylene-d2, 988 cm-1; ethylene-d1, 943 cm-1; ethylene-d0, 949 cm-1. (The various deuterated ethylenes and acetylenes are designated by a lowercase d and subscript to indicate the number, but not the position, of deuterium atoms in the molecule.) Mass spectrometry coupled to a gas chromatograph system has been used to assist in quantitation of the substrate and product distributions. Significant amounts of trans-ethylene-d2 were produced by both wild-type and nifV mutant K. pneumoniae. Less of this product was observed with the soybean system. PMID:2655535

  1. Photo- and heterotrophic nitrogenase activity by the cyano-bacterium Nostoc in symbiosis with the bryophyte Anthoceros

    SciTech Connect

    Steinberg, N.A.; Meeks, J.C.

    1987-04-01

    In symbiosis with Anthoceros, Nostoc is thought to do little or no photosynthesis. However, light-dependent /sup 14/CO/sub 2/ fixation by symbiotic Nostoc, freshly isolated from pure cultures of the reconstituted Anthoceros-Nostoc association, was 16% of that by free-living Nostoc. A DCMU-resistant mutant of Nostoc was isolated that fixed CO/sub 2/ at rates comparable to wild-type in both symbiotic and free-living growth states. To determine if symbiotic Nostoc can use its photosynthate directly to fix nitrogen, acetylene reduction by Anthoceros associations reconstituted with wild-type Nostoc was compared to associations with the DCMU-resistant mutant. In wild-type Anthoceros-Nostoc acetylene reduction was inhibited 97% by 5 ..mu..M DCMU, while inhibition of the DCMU-resistant Nostoc association was only 63%. Additions of glucose, fructose, maltose or sucrose to wild-type associations completely restored DCMU-inhibited acetylene reduction in the light. Acetylene reduction in the dark was stimulated by glucose, attaining 84% of the uninhibited light-dependent value. The authors conclude that symbiotic Nostoc maintains a pool of photosynthate which supports nitrogenase activity. The pool can also be supplemented from plant sources.

  2. Host co-factors of the retrovirus-like transposon Ty1

    PubMed Central

    2012-01-01

    Background Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. Results To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. Conclusion Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are

  3. Targeted cofactor quantification in metabolically engineered E. coli using solid phase extraction and hydrophilic interaction liquid chromatography-mass spectrometry.

    PubMed

    Li, Zhucui; Yang, Afang; Li, Yujing; Liu, Pingping; Zhang, Zhidan; Zhang, Xueli; Shui, Wenqing

    2016-03-01

    Quantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE-HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE-HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD(+) kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells. Copyright © 2016. Published by Elsevier B.V.

  4. Structural Characterization of CO-Inhibited Mo-Nitrogenase by Combined Application of Nuclear Resonance Vibrational Spectroscopy, Extended X-ray Absorption Fine Structure, and Density Functional Theory: New Insights into the Effects of CO Binding and the Role of the Interstitial Atom

    DOE PAGES

    Scott, Aubrey D.; Pelmenschikov, Vladimir; Guo, Yisong; ...

    2014-10-02

    The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm–1 mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm–1, additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by 13CO isotope shifts). The EXAFS for wild-type N2ase showsmore » evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal ₋CO and a partially reduced ₋CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational “shake” modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. We discuss implications for the CO and N2 reactivity of N2ase.« less

  5. Structural characterization of CO-inhibited Mo-nitrogenase by combined application of nuclear resonance vibrational spectroscopy, extended X-ray absorption fine structure, and density functional theory: new insights into the effects of CO binding and the role of the interstitial atom.

    PubMed

    Scott, Aubrey D; Pelmenschikov, Vladimir; Guo, Yisong; Yan, Lifen; Wang, Hongxin; George, Simon J; Dapper, Christie H; Newton, William E; Yoda, Yoshitaka; Tanaka, Yoshihito; Cramer, Stephen P

    2014-11-12

    The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm(-1) mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm(-1), additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by (13)CO isotope shifts). The EXAFS for wild-type N2ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal -CO and a partially reduced -CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational "shake" modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N2 reactivity of N2ase are discussed.

  6. Variable motif utilization in homeotic selector (Hox)-cofactor complex formation controls specificity.

    PubMed

    Lelli, Katherine M; Noro, Barbara; Mann, Richard S

    2011-12-27

    Homeotic selector (Hox) proteins often bind DNA cooperatively with cofactors such as Extradenticle (Exd) and Homothorax (Hth) to achieve functional specificity in vivo. Previous studies identified the Hox YPWM motif as an important Exd interaction motif. Using a comparative approach, we characterize the contribution of this and additional conserved sequence motifs to the regulation of specific target genes for three Drosophila Hox proteins. We find that Sex combs reduced (Scr) uses a simple interaction mechanism, where a single tryptophan-containing motif is necessary for Exd-dependent DNA-binding and in vivo functions. Abdominal-A (AbdA) is more complex, using multiple conserved motifs in a context-dependent manner. Lastly, Ultrabithorax (Ubx) is the most flexible, in that it uses multiple conserved motifs that function in parallel to regulate target genes in vivo. We propose that using different binding mechanisms with the same cofactor may be one strategy to achieve functional specificity in vivo.

  7. Ca cofactor of the water-oxidation complex: Evidence for a Mn/Ca heteronuclear cluster

    SciTech Connect

    Cinco, Roehl M.; Robblee, John H.; Messinger, Johannes; Fernandez, Carmen; McFarlane, Karen L.; Pizarro, Shelly A.; Sauer, Ken; Yachandra, Vittal K.

    2001-07-25

    Calcium and chloride are necessary cofactors for the proper function of the oxygen-evolving complex (OEC) of Photosystem II (PS II). Located in the thylakoid membranes of green plants, cyanobacteria and algae, PS II and the OEC catalyze the light-driven oxidation of water into dioxygen (released into the biosphere), protons and electrons for carbon fixation. The actual chemistry of water oxidation is performed by a cluster of four manganese atoms, along with the requisite cofactors Ca{sup 2+} and Cl{sup -}. While the Mn complex has been extensively studied by X-ray absorption techniques, comparatively less is known about the Ca{sup 2+} cofactor. The fewer number of studies on the Ca{sup 2+} cofactor have sometimes relied on substituting the native cofactor with strontium or other metals, and have stirred some debate about the structure of the binding site. past efforts using Mn EXAFS on Sr-substituted PSII are suggestive of a close link between the Mn cluster and Sr, within 3.5 {angstrom}. The most recent published study using Sr EXAFS on similar samples confirms this finding of a 3.5 {angstrom} distance between Mn and Sr. This finding was base3d on a second Fourier peak (R {approx} 3 {angstrom}) in the Sr EXAFS from functional samples, but is absent from inactive, hydroxylamine-treated PS II. This Fourier peak II was found to fit best to two Mn at 3.5 {angstrom} rather than lighter atoms (carbon). Nevertheless, other experiments have given contrary results. They wanted to extend the technique by using polarized Sr EXAFS on layered Sr-substituted samples, to provide important angle information. Polarized EXAFS involves collecting spectra for different incident angles ({theta}) between the membrane normal of the layered sample and the X-ray electric field vector. Dichroism in the EXAFS can occur, depending on how the particular absorber-backscatterer (A-B) vector is aligned with the electric field. Through analysis of the dichroism, they extract the average number

  8. Structure of a bacterial microcompartment shell protein bound to a cobalamin cofactor

    PubMed Central

    Thompson, Michael C.; Crowley, Christopher S.; Kopstein, Jeffrey; Bobik, Thomas A.; Yeates, Todd O.

    2014-01-01

    The EutL shell protein is a key component of the ethanolamine-utilization microcompartment, which serves to compartmentalize ethanolamine degradation in diverse bacteria. The apparent function of this shell protein is to facilitate the selective diffusion of large cofactor molecules between the cytoplasm and the lumen of the microcompartment. While EutL is implicated in molecular-transport phenomena, the details of its function, including the identity of its transport substrate, remain unknown. Here, the 2.1 Å resolution X-ray crystal structure of a EutL shell protein bound to cobalamin (vitamin B12) is presented and the potential relevance of the observed protein–ligand interaction is briefly discussed. This work represents the first structure of a bacterial microcompartment shell protein bound to a potentially relevant cofactor molecule. PMID:25484204

  9. Menaquinone-7 Is Specific Cofactor in Tetraheme Quinol Dehydrogenase CymA

    PubMed Central

    McMillan, Duncan G. G.; Marritt, Sophie J.; Butt, Julea N.; Jeuken, Lars J. C.

    2012-01-01

    Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions. PMID:22393052

  10. The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria.

    PubMed

    Leimkühler, Silke; Wuebbens, Margot M; Rajagopalan, K V

    2011-05-01

    Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center.

  11. The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria

    PubMed Central

    Leimkühler, Silke; Wuebbens, Margot M.; Rajagopalan, K.V.

    2010-01-01

    Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center. PMID:21528011

  12. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

    PubMed Central

    Cruz-Reyes, Jorge; Mooers, Blaine H.M.; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    2016-01-01

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. PMID:27540585

  13. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor.

    PubMed

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven

    2011-08-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

  14. Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

    PubMed Central

    Singh, Appu Kumar; Mondal, Alok K.; Kumaran, S.

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. PMID:23049810

  15. Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

    PubMed

    Biswas, Dipanwita; Pandya, Vaibhav; Singh, Appu Kumar; Mondal, Alok K; Kumaran, S

    2012-01-01

    Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

  16. Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2

    PubMed Central

    Fourmann, Jean-Baptiste; Tauchert, Marcel J.; Ficner, Ralf

    2017-01-01

    Abstract The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly. PMID:27923990

  17. Thermodynamic analysis of interactions between cofactor and neuronal nitric oxide synthase.

    PubMed

    Sanae, Ryuhei; Kurokawa, Fumiaki; Oda, Masayuki; Ishijima, Sumio; Sagami, Ikuko

    2011-03-15

    The thermodynamics of cofactor binding to the isolated reductase domain (Red) of nNOS and its mutants have been studied by isothermal titration calorimetry. The NADP(+) and 2',5'-ADP binding stoichiometry to Red were both 1:1, consistent with a one-site kinetic model instead of a two-site model. The binding constant (K(D) = 71 nM) and the large heat capacity change (ΔC(p) = -440 cal mol(-1) K(-1)) for 2',5'-ADP were remarkably different from those for NADP(+) (1.7 μM and -140 cal mol(-1) K(-1), respectively). These results indicate that the nicotinamide moiety as well as the adenosine moiety has an important role in binding to nNOS. They also suggest that the thermodynamics of the conformational change in Red caused by cofactor binding are significantly different from the conformational changes that occur in cytochrome c reductase, in which the nicotinamide moiety of the cofactor is not essential for binding. Analysis of the deletion mutant of the autoinhibitory helix (RedΔ40) revealed that the deletion resulted in a decrease in the binding affinity of 2',5'-ADP with more unfavorable enthalpy gain. In the case of RedCaM, which contains a calmodulin (CaM) binding site, the presence of Ca(2+)/CaM caused a 6.7-fold increase in the binding affinity for 2',5'-ADP that was mostly due to the favorable entropy change. These results are consistent with a model in which Ca(2+)/CaM induces a conformational change in NOS to a flexible "open" form from a "closed" form that locked by cofactor binding, and this change facilitates the electron transfer required for catalysis.

  18. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor

    PubMed Central

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung

    2011-01-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function. PMID:21720211

  19. Stress and Activity of Molybdenum-Containing Complex (Molybdenum Cofactor) in Winter Wheat Seeds

    PubMed Central

    Vunkova-Radeva, Reneta; Schiemann, Johan; Mendel, Ralf-Reiner; Salcheva, Galitona; Georgieva, Damyana

    1988-01-01

    Molybdenum, applied in vivo, restored the damage from low temperature with winter wheat (Triticum aestivum, var “Sadovo 1”) grown on acid soil and, in addition, sharply increased productivity (G Salcheva, D Georgieva, 1982; G Salcheva et al., 1977, 1979). Two fractions with molybdenum-cofactor activity in seeds were detected. One of them has a molecular weight of about 230 kilodaltons corresponding to xanthine oxidase activity and leaf nitrate reductase activity. The other has a molecular weight of about 60 kilodaltons. The ratio between the molybdenum-cofactor activity of these fractions was different in `mother' seeds used in the experiment, in seeds obtained from the damaged plants, and in seeds obtained from the damaged plants restored by in vivo molybdenum addition. Every one of these fractions consisted of several components in which molybdenum-cofactor activity and stability in vitro was different. We suggest that plants store molybdenum as molybdenum carriers in these low molecular weight fractions. Images Fig. 2 PMID:16666178

  20. Developmental expression patterns of candidate co-factors for vertebrate Six family transcription factors

    PubMed Central

    Neilson, Karen M.; Pignoni, Francesca; Yan, Bo; Moody, Sally A.

    2010-01-01

    Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by co-factor proteins. Two Six genes and one co-factor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for about half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila co-factor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes. PMID:21089078

  1. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    SciTech Connect

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  2. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases

    PubMed Central

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J.; Kaput, Jim; Priami, Corrado

    2016-01-01

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900’s at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases. PMID:26777674

  3. Intracellular Trafficking of the Pyridoxal Cofactor. Implications for Health and Metabolic Disease*

    PubMed Central

    Whittaker, James W.

    2016-01-01

    The importance of the vitamin B6-derived pyridoxal cofactor for human health has been established through more than 70 years of intensive biochemical research, revealing its fundamental roles in metabolism. B6 deficiency, resulting from nutritional limitation or impaired uptake from dietary sources, is associated with epilepsy, neuromuscular disease and neurodegeneration. Hereditary disorders of B6 processing are also known, and genetic defects in pathways involved in transport of B6 into the cell and its transformation to the pyridoxal-5′-phosphate enzyme cofactor can contribute to cardiovascular disease by interfering with homocysteine metabolism and the biosynthesis of vasomodulatory polyamines. Compared to the processes involved in cellular uptake and processing of the B6 vitamers, trafficking of the PLP cofactor across intracellular membranes is very poorly understood, even though the availability of PLP within subcellular compartments (particularly the mitochondrion) may have important health implications. The aim of this review is to concisely summarize the state of current knowledge of intracellular trafficking of PLP and to identify key directions for future research. PMID:26619753

  4. Caenorhabditis elegans UBX cofactors for CDC-48/p97 control spermatogenesis.

    PubMed

    Sasagawa, Yohei; Yamanaka, Kunitoshi; Saito-Sasagawa, Yuko; Ogura, Teru

    2010-12-01

    UBX (ubiquitin regulatory X) domain-containing proteins act as cofactors for CDC-48/p97. CDC-48/p97 is essential for various cellular processes including retro-translocation in endoplasmic reticulum-associated degradation, homotypic membrane fusion, nuclear envelope assembly, degradation of ubiquitylated proteins, and cell cycle progression. CDC-48/p97-dependent processes are determined by differential binding of cofactors including UBX proteins, but the cellular functions of UBX proteins have not yet been elucidated, especially in multicellular organisms. Therefore, we investigated the functions of UBX family members using Caenorhabditis elegans, which expresses six UBX proteins, UBXN-1 to UBXN-6. All six UBXN proteins directly interacted with CDC-48.1 and CDC-48.2, and simultaneous knockdown of the expression of three genes, ubxn-1, ubxn-2 and ubxn-3, induced embryonic lethal and sterile phenotypes, but knockdown of either one or two did not. The sterile worms had a feminized germ-line phenotype, producing oocytes but no sperm. UBXN-1, UBXN-2 and UBXN-3 colocalized with CDC-48 in spermatocytes but not mature sperm. TRA-1A, which is a key factor in the sex determination pathway and inhibits spermatogenesis, accumulated in worms in which UBXN-1, UBXN-2 and UBXN-3 had been simultaneously knocked down. Taken together, these results suggest that UBXN-1, UBXN-2 and UBXN-3 are redundant cofactors for CDC-48/p97 and control spermatogenesis via the degradation of TRA-1A.

  5. Substrate recognition and catalysis by the cofactor-independent dioxygenase DpgC.

    PubMed

    Fielding, Elisha N; Widboom, Paul F; Bruner, Steven D

    2007-12-11

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC [Widboom, P. W., Fielding, E. N., Liu, Y., and Bruner, S. D. (2007) Nature 447, 342-345] confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  6. A Live Zebrafish-Based Screening System for Human Nuclear Receptor Ligand and Cofactor Discovery

    PubMed Central

    Tiefenbach, Jens; Moll, Pamela R.; Nelson, Meryl R.; Hu, Chun; Baev, Lilia; Kislinger, Thomas; Krause, Henry M.

    2010-01-01

    Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors. PMID:20339547

  7. Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

    PubMed Central

    Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

    2013-01-01

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

  8. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases.

    PubMed

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J; Kaput, Jim; Priami, Corrado

    2016-01-18

    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900's at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases.

  9. Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

    PubMed Central

    Cuzic, Simona; Hartmann, Roland K.

    2005-01-01

    We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)63+ and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)63+ substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance. PMID:15867194

  10. Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa*

    PubMed Central

    Ringel, Phillip; Krausze, Joern; van den Heuvel, Joop; Curth, Ute; Pierik, Antonio J.; Herzog, Stephanie; Mendel, Ralf R.; Kruse, Tobias

    2013-01-01

    Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process. PMID:23539622

  11. The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria.

    PubMed

    Yokoyama, Kenichi; Leimkühler, Silke

    2015-06-01

    The biosynthesis of the molybdenum cofactor (Moco) has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the assembly of molybdoenzymes and the biosynthesis of FeS clusters has been identified in the recent years: 1) the synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) the sulfurtransferase for the dithiolene group in Moco is also involved in the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the addition of a sulfido-ligand to the molybdenum atom in the active site additionally involves a sulfurtransferase, and 4) most molybdoenzymes in bacteria require FeS clusters as redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Pyridoxine-dependent epilepsy with elevated urinary α-amino adipic semialdehyde in molybdenum cofactor deficiency.

    PubMed

    Struys, Eduard Alexander; Nota, Benjamin; Bakkali, Abdellatif; Al Shahwan, Saad; Salomons, Gajja Sophi; Tabarki, Brahim

    2012-12-01

    α-Amino adipic semialdehyde (α-AASA) accumulates in body fluids from patients with pyridoxine-dependent epilepsy because of mutations in antiquitin (ALDH7A1) and serves as the biomarker for this condition. We have recently found that the urinary excretion of α-AASA was also increased in molybdenum cofactor and sulfite oxidase deficiencies. The seizures in pyridoxine-dependent epilepsy are caused by lowered cerebral levels of pyridoxal-5-phosphate (PLP), the bioactive form of pyridoxine (vitamin B(6)), which can be corrected by the supplementation of pyridoxine. The nonenzymatic trapping of PLP by the cyclic form of α-AASA is causative for the lowered cerebral PLP levels. We describe 2 siblings with clinically evident pyridoxine-responsive seizures associated with increased urinary excretion of α-AASA. Subsequent metabolic investigations revealed several metabolic abnormities, all indicative for molybdenum cofactor deficiency. Molecular investigations indeed revealed a known homozygous mutation in the MOCS2 gene. Based upon the clinically evident pyridoxine-responsive seizures in these 2 siblings, we recommend considering pyridoxine supplementation to patients affected with molybdenum cofactor or sulfite oxidase deficiencies.

  13. Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

    PubMed Central

    Li, Yajing; van der Est, Art; Lucas, Marie Gabrielle; Ramesh, V. M.; Gu, Feifei; Petrenko, Alexander; Lin, Su; Webber, Andrew N.; Rappaport, Fabrice; Redding, Kevin

    2006-01-01

    Photosystem I has two branches of cofactors down which light-driven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (FX) is described by two kinetic components with decay times of ≈20 and ≈200 ns, which have been proposed to represent ET from PhQB and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to FX. The mutation near ec3A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors. PMID:16467143

  14. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

    PubMed Central

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo

    2016-01-01

    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle. PMID:27432985

  15. Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

    PubMed

    Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

    2015-06-15

    Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site. © The Authors Journal Compilation © 2015 Biochemical Society.

  16. Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

    PubMed

    Park, Jong Hyun; Lee, Sahng Ha; Cha, Gun Su; Choi, Da Som; Nam, Dong Heon; Lee, Jae Hyung; Lee, Jung-Kul; Yun, Chul-Ho; Jeong, Ki Jun; Park, Chan Beum

    2015-01-12

    Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17β-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches.

    PubMed

    Li, Yajing; van der Est, Art; Lucas, Marie Gabrielle; Ramesh, V M; Gu, Feifei; Petrenko, Alexander; Lin, Su; Webber, Andrew N; Rappaport, Fabrice; Redding, Kevin

    2006-02-14

    Photosystem I has two branches of cofactors down which light-driven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (FX) is described by two kinetic components with decay times of approximately 20 and approximately 200 ns, which have been proposed to represent ET from PhQB and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to F(X). The mutation near ec3A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors.

  18. The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response.

    PubMed

    Staudigl, Michael; Gersting, Søren W; Danecka, Marta K; Messing, Dunja D; Woidy, Mathias; Pinkas, Daniel; Kemter, Kristina F; Blau, Nenad; Muntau, Ania C

    2011-07-01

    The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.

  19. Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

    PubMed

    Ringel, Phillip; Krausze, Joern; van den Heuvel, Joop; Curth, Ute; Pierik, Antonio J; Herzog, Stephanie; Mendel, Ralf R; Kruse, Tobias

    2013-05-17

    Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

  20. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics.

    PubMed

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo

    2016-07-26

    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.

  1. Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

    PubMed Central

    Huijbers, Mieke M. E.; Martínez-Júlvez, Marta; Westphal, Adrie H.; Delgado-Arciniega, Estela; Medina, Milagros; van Berkel, Willem J. H.

    2017-01-01

    Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered. PMID:28256579

  2. Distinct structural features of the. cap alpha. and. beta. subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

    SciTech Connect

    Wang, S.Z.; Chen, J.S.; Johnson, J.L.

    1988-04-19

    Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding ..cap alpha.. and ..beta.. subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated ..beta.. subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (M/sub r/ 50,115) whose amino-terminal region is about 50 resides shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp ..cap alpha.. subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the ..cap alpha.. and ..beta.. subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine resides, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.

  3. Probing the MgATP-Bound Conformation of the Nitrogenase Fe Protein By Solution Small-Angle X-Ray Scattering

    SciTech Connect

    Sarma, R.; Mulder, D.W.; Brecht, E.; Szilagyi, R.K.; Seefeldt, L.C.; Tsuruta, H.; Peters, J.W.; /Montana State U. /SLAC, SSRL /Utah State U.

    2009-04-30

    The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small-angle X-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single-amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits, resulting in an overall elongated structure relative to the more compact structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein largely on the basis of the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATP-bound state. In this work, SAXS studies reveal that the large-scale conformational differences between the native Fe protein and the variant observed by X-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.

  4. Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

    PubMed Central

    Elsen, Sylvie; Dischert, Wanda; Colbeau, Annette; Bauer, Carl E.

    2000-01-01

    Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H2 oxidation, as well as the energy-demanding processes of N2 fixation and CO2 assimilation. PMID:10781552

  5. Nitrogenase of Klebsiella pneumoniae. Inhibition of acetylene reduction by magnesium ion explained by the formation of an inactive dimagnesium–adenosine triphosphate complex

    PubMed Central

    Thorneley, Roger N. F.; Willison, Keith R.

    1974-01-01

    Acetylene-reducing activity of purified nitrogenase from Klebsiella pneumoniae was studied over a range of ATP and Mg2+ concentrations at 15°C, pH7.8. Inhibition at Mg2+ concentrations of 2.5–30mm was due to the formation of the inactive complex, Mg2ATP. At higher Mg2+ concentrations an additional inhibitory effect was observed. The results were consistent with a MgATP complex being the active substrate with an apparent Km(MgATP)=0.4mm. PMID:4618775

  6. Colonization and nitrogenase activity of Triticum aestivum (cv. Baccross and Mahdavi) to the dual inoculation with Azospirillum brasilense and Rhizobium meliloti plus 2,4-D.

    PubMed

    Mehry, Askary; Akbar, Mostajeran; Giti, Emtiazi

    2008-06-15

    The potential enhancement of root colonization and nitrogenase activity of wheat cultivars (Baccross and Mahdavi) was studied with application of two Azospirillum brasilense strains (native and Sp7) co-inoculated with two Rhizobium meliloti strains (native and DSMZ 30135). The results indicated that the colonization was different due to the strains and cultivars of wheat were used. Native A. brasilense colonized wheat root better than Sp7 strain. However, Baccross cv. reacted better with native Azospirillum compared to Mahdavi cv. which reacted better with Sp7. When plants inoculated with dual inoculants (SP7 with standard Rhizobium), the colonization of Azospirillum were increased significantly (from 1.67 x 10(5) to 22 x 10(5) cfu g(-1) FW for Baccras cv. and 3.67 x 10(5) to 26 x 10(5) cfu g(-1) FW for Mahdavi cultivar). When the standard Rhizobium as co-inoculants changed to the native Rhizobium, the colonization of Azospirillum was higher when compared to the single inoculants but was almost the same when compared to the standard Rhizobium. When the standard or native strains of Rhizobium used as single inoculation of wheat roots, the number of Rhizobium in the wheat roots were not changed significantly. However, when plants co-inoculated with Rhizobium and Azospirillum, the colonization of Rhizobium was increased. Co-inoculation of standard strain of R. melilot with A. brasilense Sp7 showed that the colonization of Rhizobium were increased from 0.67 x 10(5) to 21 x 10(5) cfu g(-1) FW for Baccross cv. and 0.33 x 10(5) to 18 x 10(5) cfu g(-1) FW for Mahdavi cv. This behavior was the same when inoculation of Rhizobium was happened with the native one. In dual inoculation, the highest nitrogenase activity was measured in combination of the local strains (native A. brasilense with the native R. meliloti) and the lower one belongs to the combination of standard strains (Sp7 with standard R. meliloti). The difference in nirtogenase activity for different cultivars of

  7. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    NASA Astrophysics Data System (ADS)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  8. Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding.

    PubMed

    Rao, Maya V; Williams, Dewight R; Cocklin, Simon; Loll, Patrick J

    2017-09-22

    p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum associated degradation (ERAD). However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif (VBM) interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C-terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  9. Feline leukemia virus T entry is dependent on both expression levels and specific interactions between cofactor and receptor.

    PubMed

    Cheng, Heather H; Anderson, Maria M; Overbaugh, Julie

    2007-03-01

    Feline leukemia virus (FeLV) subgroup T uses both a multiple membrane-spanning receptor, FePit1, and a soluble cofactor, FeLIX, to enter feline cells. FeLIX is expressed from endogenous FeLV-related sequence and resembles the receptor binding domain (RBD) of the viral envelope protein. It remains unclear whether FeLV-T receptor activity requires specific residues within FePit1 and FeLIX and/or a threshold level of receptor/cofactor expression. To address this, we examined FeLV-T infection of cells expressing variable levels of FePit1 and other gammaretroviral receptors in the presence of variable amounts of soluble cofactor, either RBD or the envelope surface subunit (SU). Cofactor-receptor pairs fall into three groups with regard to mediating FeLV-T infection: those that are efficient at all concentrations tested, such as FePit1 and FeLIX; those requiring high expression of both cofactor and receptor; and those that are non-functional as receptors even at high expression. This suggests that both expression levels and specific interactions with receptor and cofactor are critical for mediating entry of FeLV-T.

  10. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

    PubMed

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

  11. The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme.

    PubMed

    Bingaman, Jamie L; Zhang, Sixue; Stevens, David R; Yennawar, Neela H; Hammes-Schiffer, Sharon; Bevilacqua, Philip C

    2017-04-01

    RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.

  12. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors

    PubMed Central

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product. PMID:26441891

  13. Endogenous Ethylene Production Is a Potential Problem in the Measurement of Nitrogenase Activity Associated with Excised Corn and Sorghum Roots 1

    PubMed Central

    Sloger, Charles; van Berkum, Peter

    1988-01-01

    Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots. PMID:16666249

  14. Expression and Association of Group IV Nitrogenase NifD and NifH Homologs in the Non-Nitrogen-Fixing Archaeon Methanocaldococcus jannaschii▿ †

    PubMed Central

    Staples, Christopher R.; Lahiri, Surobhi; Raymond, Jason; Von Herbulis, Lindsay; Mukhophadhyay, Biswarup; Blankenship, Robert E.

    2007-01-01

    Using genomic analysis, researchers previously identified genes coding for proteins homologous to the structural proteins of nitrogenase (J. Raymond, J. L. Siefert, C. R. Staples, and R. E. Blankenship, Mol. Biol. Evol. 21:541-554, 2004). The expression and association of NifD and NifH nitrogenase homologs (named NflD and NflH for “Nif-like” D and H, respectively) have been detected in a non-nitrogen-fixing hyperthermophilic methanogen, Methanocaldococcus jannaschii. These homologs are expressed constitutively and do not appear to be directly involved with nitrogen metabolism or detoxification of compounds such as cyanide or azide. The NflH and NflD proteins were found to interact with each other, as determined by bacterial two-hybrid studies. Upon immunoisolation, NflD and NflH copurified, along with three other proteins whose functions are as yet uncharacterized. The apparent presence of genes coding for NflH and NflD in all known methanogens, their constitutive expression, and their high sequence similarity to the NifH and NifD proteins or the BchL and BchN/BchB proteins suggest that NflH and NflD participate in an indispensable and fundamental function(s) in methanogens. PMID:17660283

  15. Multi-omic dynamics associate oxygenic photosynthesis with nitrogenase-mediated H2 production in Cyanothece sp. ATCC 51142

    SciTech Connect

    Bernstein, Hans C.; Charania, Moiz A.; McClure, Ryan S.; Sadler, Natalie C.; Melnicki, Matthew R.; Hill, Eric A.; Markillie, Lye Meng; Nicora, Carrie D.; Wright, Aaron T.; Romine, Margaret F.; Beliaev, Alexander S.

    2015-11-03

    This study combines transcriptomic and proteomic profiling to provide new insights on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in the model cyanobacterium, Cyanothece sp. ATCC 51142. To date, the proposed mechanisms used to describe the energy metabolism processes that support H2 production in Cyanothece 51142 have assumed that ATP and reductant requirements are derived solely from glycogen oxidation and/or cyclic-electron flow around photosystem I. The results from this study present and test an alternative hypothesis by showing that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the likely role of photocatalytic H2O oxidation as a major participating process.

  16. Analogue-resistant mutants of Azotobacter chroococcum derepressed for nitrogenase activity and early ammonia excretion having potential as inoculants for cereal crops.

    PubMed

    Lakshminarayana, K; Shukla, B; Sindhu, S S; Kumari, P; Narula, N; Sheoran, R K

    2000-04-01

    Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.

  17. Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora.

    PubMed

    Yoch, D C; Whiting, G J

    1986-01-01

    The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.

  18. Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum.

    PubMed Central

    Chen, K C; Chen, J S; Johnson, J L

    1986-01-01

    The structural gene (nifH1) encoding the nitrogenase iron protein of Clostridium pasteurianum has been cloned and sequenced. It is located on a 4-kilobase EcoRI fragment (cloned into pBR325) that also contains a portion of nifD and another nifH-like sequence (nifH2). C. pasteurianum nifH1 encodes a polypeptide (273 amino acids) identical to that of the isolated iron protein, indicating that the smaller size of the C. pasteurianum iron protein does not result from posttranslational processing. The 5' flanking region of nifH1 or nifH2 does not contain the nif promoter sequences found in several gram-negative bacteria. Instead, a sequence resembling the Escherichia coli consensus promoter (TTGACA-N17-TATAAT) is present before C. pasteurianum nifH2, and a TATAAT sequence is present before C pasteurianum nifH1. Codon usage in nifH1, nifH2, and nifD (partial) is very biased. A preference for A or U in the third position of the codons is seen. nifH2 could encode a protein of 272 amino acid residues, which differs from the iron protein (nifH1 product) in 23 amino acid residues (8%). Another nifH-like sequence (nifH3) is located on a nonadjacent EcoRI fragment and has been partially sequenced. C. pasteurianum nifH2 and nifH3 may encode proteins having several amino acids that are conserved in other proteins but not in C. pasteurianum iron protein, suggesting a possible role for the multiple nifH-like sequences of C. pasteurianum in the evolution of nifH. Among the nine sequenced iron proteins, only the C. pasteurianum protein lacks a conserved lysine residue which is near the extended C terminus of the other iron proteins. The absence of this positive charge in the C. pasteurianum iron protein might affect the cross-reactivity of the protein in heterologous systems. Images PMID:3457003

  19. Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

    PubMed Central

    Sanli, Gulsah; Banta, Scott; Anderson, Stephen; Blaber, Michael

    2004-01-01

    Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified. PMID:14718658

  20. Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors.

    PubMed

    Bou-Torrent, Jordi; Toledo-Ortiz, Gabriela; Ortiz-Alcaide, Miriam; Cifuentes-Esquivel, Nicolas; Halliday, Karen J; Martinez-García, Jaime F; Rodriguez-Concepcion, Manuel

    2015-11-01

    Carotenoids are photosynthetic pigments essential for the protection against excess light. During deetiolation, their production is regulated by a dynamic repression-activation module formed by PHYTOCHROME-INTERACTING FACTOR1 (PIF1) and LONG HYPOCOTYL5 (HY5). These transcription factors directly and oppositely control the expression of the gene encoding PHYTOENE SYNTHASE (PSY), the first and main rate-determining enzyme of the carotenoid pathway. Antagonistic modules also regulate the responses of deetiolated plants to vegetation proximity and shade (i.e. to the perception of far-red light-enriched light filtered through or reflected from neighboring plants). These responses, aimed to adapt to eventual shading from plant competitors, include a reduced accumulation of carotenoids. Here, we show that PIF1 and related photolabile PIFs (but not photostable PIF7) promote the shade-triggered decrease in carotenoid accumulation. While HY5 does not appear to be required for this process, other known PIF antagonists were found to modulate the expression of the Arabidopsis (Arabidopsis thaliana) PSY gene and the biosynthesis of carotenoids early after exposure to shade. In particular, PHYTOCHROME-RAPIDLY REGULATED1, a transcriptional cofactor that prevents the binding of true transcription factors to their target promoters, was found to interact with PIF1 and hence directly induce PSY expression. By contrast, a change in the levels of the transcriptional cofactor LONG HYPOCOTYL IN FAR RED1, which also binds to PIF1 and other PIFs to regulate shade-related elongation responses, did not impact PSY expression or carotenoid accumulation. Our data suggest that the fine-regulation of carotenoid biosynthesis in response to shade relies on specific modules of antagonistic transcriptional factors and cofactors.