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Sample records for nitrogenase femo cofactor

  1. Nitrogenase-catalyzed ethane production and CO-sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an alpha-subunit FeMo cofactor-binding domain.

    PubMed

    Scott, D J; Dean, D R; Newton, W E

    1992-10-01

    Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the

  2. Radical S-adenosyl-L-methionine chemistry in the synthesis of hydrogenase and nitrogenase metal cofactors.

    PubMed

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

    2015-02-13

    Nitrogenase, [FeFe]-hydrogenase, and [Fe]-hydrogenase enzymes perform catalysis at metal cofactors with biologically unusual non-protein ligands. 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. This minireview presents and discusses the current state of knowledge of the radical S-adenosylmethionine enzymes required for synthesis of these remarkable metal cofactors.

  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

    2015-11-01

    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.

  4. 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

  5. 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.

  6. A journey into the active center of nitrogenase.

    PubMed

    Hu, Yilin; Ribbe, Markus W

    2014-08-01

    Nitrogenase catalyzes the reduction of N2 to NH3, a key step in the global nitrogen cycle. This article describes our journey toward the definition of a complete molecular structure of the active site of nitrogenase, with an emphasis on the discovery of the interstitial carbide and the radical SAM-dependent insertion of this atom into the active FeMo cofactor site of nitrogenase.

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

    PubMed

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

    2015-01-19

    Nitrogenase cofactors can be extracted into an organic solvent to catalyze the reduction of cyanide (CN(-)), carbon monoxide (CO), and carbon dioxide (CO2) without using adenosine triphosphate (ATP), when samarium(II) iodide (SmI2) and 2,6-lutidinium triflate (Lut-H) are employed as a reductant and a proton source, respectively. Driven by SmI2, the cofactors catalytically reduce CN(-) or CO to C1-C4 hydrocarbons, and CO2 to CO and C1-C3 hydrocarbons. The C-C coupling from CO2 indicates a unique Fischer-Tropsch-like reaction with an atypical carbonaceous substrate, whereas the catalytic turnover of CN(-), CO, and CO2 by isolated cofactors suggests the possibility to develop nitrogenase-based electrocatalysts for the production of hydrocarbons from these carbon-containing compounds.

  8. 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

  9. Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor.

    PubMed Central

    Pau, R N; Eldridge, M E; Lowe, D J; Mitchenall, L A; Eady, R R

    1993-01-01

    Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components. PMID:8392330

  10. On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase

    PubMed Central

    Yang, Zhi-Yong; Khadka, Nimesh; Lukoyanov, Dmitriy; Hoffman, Brian M.; Dean, Dennis R.; Seefeldt, Lance C.

    2013-01-01

    Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 bound to FeMo-cofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H+ with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism. PMID:24062454

  11. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

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

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  12. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

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

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  13. 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

  14. In vitro synthesis of the iron-molybdenum cofactor of nitrogenase.

    PubMed Central

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

    1986-01-01

    Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae. PMID:3006060

  15. COMPARATIVE ASSESSMENT OF THE COMPOSITION AND CHARGE STATE OF NITROGENASE FeMo-COFACTOR

    PubMed Central

    Harris, Travis V.; Szilagyi, Robert K.

    2011-01-01

    A significant limitation in our understanding of the molecular mechanism of biological nitrogen fixation is the uncertain composition of the FeMo-cofactor (FeMo-co) of nitrogenase. In this study we present a systematic, density functional theory-based evaluation of spin coupling schemes, iron oxidation states, ligand protonation states, and interstitial ligand composition using a wide range of experimental criteria. The employed functionals and basis sets were validated with molecular orbital information from X-ray absorption spectroscopic data of relevant iron-sulfur clusters. Independently from the employed level of theory, the electronic structure with the greatest number of antiferromagnetic interactions corresponds to the lowest energy state for a given charge and oxidation state distribution of the iron ions. The relative spin state energies of resting and oxidized FeMo-co already allowed the exclusion of certain iron oxidation state distributions and interstitial ligand compositions. Geometry optimized FeMo-co structures of several models further eliminated additional states and compositions, while reduction potentials indicated a strong preference for the most likely charge state of FeMo-co. Mössbauer and ENDOR parameter calculations were found to be remarkably dependent on the employed training set, density functional and basis set. Overall, we found that a more oxidized [MoIV-2FeII-5FeIII-9S2−-C4−] composition with a hydroxyl-protonated homocitrate ligand satisfies all of the available experimental criteria, and is thus favored over the currently preferred composition of [MoIV-4FeII-3FeIII-9S2−-N3−] from the literature. PMID:21545160

  16. Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states.

    PubMed

    Newton, W E; Gheller, S F; Feldman, B J; Dunham, W R; Schultz, F A

    1989-02-01

    Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.

  17. 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.

  18. Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene.

    PubMed

    Fisher, K; Dilworth, M J; Kim, C H; Newton, W E

    2000-03-21

    Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to

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

    PubMed

    Wilcoxen, Jarett; Arragain, Simon; Scandurra, Alessandro A; Jimenez-Vicente, Emilio; Echavarri-Erasun, Carlos; Pollmann, Stephan; Britt, R David; Rubio, Luis M

    2016-06-22

    NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed. PMID:27268267

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

    PubMed

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

    2016-09-01

    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

  1. 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

  2. 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.

  3. 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

    2016-10-01

    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.

  4. A vanadium and iron cluster accumulates on VnfX during iron-vanadium-cofactor synthesis for the vanadium nitrogenase in Azotobacter vinelandii.

    PubMed

    Rüttimann-Johnson, C; Staples, C R; Rangaraj, P; Shah, V K; Ludden, P W

    1999-06-18

    The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7-8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

  5. Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

    PubMed Central

    Davis, R; Lehman, L; Petrovich, R; Shah, V K; Roberts, G P; Ludden, P W

    1996-01-01

    The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane. PMID:8631723

  6. 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

  7. Classifying the metal dependence of uncharacterized nitrogenases.

    PubMed

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

    2012-01-01

    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 (N(2)) to ammonia (NH(3)) but exhibit differences in catalytic rates and substrate specificity unique to metal type. Recently, N(2) 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

  8. 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

  9. 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

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

    2014-12-01

    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 [(Δ)VO(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 2.003Å respectively, which are comparable to the VO distance (2.15Å) 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.

  10. 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

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

    2014-12-01

    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 [(Δ)VO(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 2.003Å respectively, which are comparable to the VO distance (2.15Å) 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

  11. Molybdenum L-Edge XAS Spectra of MoFe Nitrogenase

    PubMed Central

    Bjornsson, Ragnar; Delgado-Jaime, Mario U; Lima, Frederico A; Sippel, Daniel; Schlesier, Julia; Weyhermüller, Thomas; Einsle, Oliver; Neese, Frank; DeBeer, Serena

    2015-01-01

    A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco. PMID:26213424

  12. 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.

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

    PubMed

    Dance, Ian

    2015-11-01

    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

  14. 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

  15. Conserved Amino Acid Sequence Features in the α Subunits of MoFe, VFe, and FeFe Nitrogenases

    PubMed Central

    Glazer, Alexander N.; Kechris, Katerina J.

    2009-01-01

    Background This study examines the structural features and phylogeny of the α subunits of 69 full-length NifD (MoFe subunit), VnfD (VFe subunit), and AnfD (FeFe subunit) sequences. Methodology/Principal Findings The analyses of this set of sequences included BLAST scores, multiple sequence alignment, examination of patterns of covariant residues, phylogenetic analysis and comparison of the sequences flanking the conserved Cys and His residues that attach the FeMo cofactor to NifD and that are also conserved in the alternative nitrogenases. The results show that NifD nitrogenases fall into two distinct groups. Group I includes NifD sequences from many genera within Bacteria, including all nitrogen-fixing aerobes examined, as well as strict anaerobes and some facultative anaerobes, but no archaeal sequences. In contrast, Group II NifD sequences were limited to a small number of archaeal and bacterial sequences from strict anaerobes. The VnfD and AnfD sequences fall into two separate groups, more closely related to Group II NifD than to Group I NifD. The pattern of perfectly conserved residues, distributed along the full length of the Group I and II NifD, VnfD, and AnfD, confirms unambiguously that these polypeptides are derived from a common ancestral sequence. Conclusions/Significance There is no indication of a relationship between the patterns of covariant residues specific to each of the four groups discussed above that would give indications of an evolutionary pathway leading from one type of nitrogenase to another. Rather the totality of the data, along with the phylogenetic analysis, is consistent with a radiation of Group I and II NifDs, VnfD and AnfD from a common ancestral sequence. All the data presented here strongly support the suggestion made by some earlier investigators that the nitrogenase family had already evolved in the last common ancestor of the Archaea and Bacteria. PMID:19578539

  16. Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

    PubMed Central

    Lee, Chi Chung; Fay, Aaron W.; Weng, Tsu-Chien; Krest, Courtney M.; Hedman, Britt; Hodgson, Keith O.; Hu, Yilin; Ribbe, Markus W.

    2015-01-01

    Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction. PMID:26515097

  17. 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

  18. 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.…

  19. 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.

  20. Posttranslational modification of a vanadium nitrogenase

    PubMed Central

    Heiniger, Erin K; Harwood, Caroline S

    2015-01-01

    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. PMID:26097040

  1. Nitrogenase Fe protein: A molybdate/homocitrate insertase

    PubMed Central

    Hu, Yilin; Corbett, Mary C.; Fay, Aaron W.; Webber, Jerome A.; Hodgson, Keith O.; Hedman, Britt; Ribbe, Markus W.

    2006-01-01

    The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein–protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase. PMID:17062756

  2. Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

    PubMed Central

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

    2014-01-01

    All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting. PMID:25139995

  3. Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

    PubMed

    Miller, R W; Eady, R R

    1988-12-01

    A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

  4. 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.

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

    PubMed

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

    2015-08-11

    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.

  6. 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

  7. 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.

  8. 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.

  9. Structural and magnetic relaxations of mechanically alloyed Fe-Mo

    NASA Astrophysics Data System (ADS)

    Jiraskova, Y.; Bursik, J.; Turek, I.; Cizek, J.; Prochazka, I.

    2014-10-01

    The Fe-Mo sample mechanically alloyed for 250 h under air atmosphere was exposed to a series of isothermal and isochronal treatments with the aim to follow changes in the structure and magnetic properties regarding relaxations of strains and defects and stability of chemical composition. For this purpose x-ray diffraction, positron annihilation, scanning and transmission electron microscopy, and Mössbauer spectrometry were applied and supplemented by magnetic measurements. The temperatures for the magnetic studies were selected from the thermomagnetic curve of the as-prepared sample. The time interval of isothermal treatments was chosen from 0-300 min. The Mo content in the bcc-Fe(Mo) phase has substantially exceeded the equilibrium solubility limit but it has been found to decrease under the thermal treatment which was reflected by decreasing lattice parameters. The small crystallite size of approximately 10 nm in the initial state starts to grow only after a certain amount of strains induced by severe deformation, due to mechanical alloying being released. This was also reflected in the magnetic parameters. From their time dependences at selected temperatures the characteristic relaxation times were obtained and used for a calculation of the activation enthalpy of relaxation processes.

  10. Expression of the nifBfdxNnifOQ region of Azotobacter vinelandii and its role in nitrogenase activity.

    PubMed Central

    Rodríguez-Quiñones, F; Bosch, R; Imperial, J

    1993-01-01

    The nifBQ transcriptional unit of Azotobacter vinelandii has been previously shown to be required for activity of the three nitrogenase systems, Mo nitrogenase, V nitrogenase, and Fe nitrogenase, present in this organism. We studied regulation of expression and the role of the nifBQ region by means of translational beta-galactosidase fusions to each of the five open reading frames: nifB, orf2 (fdxN), orf3 (nifO), nifQ, and orf5. Expression of the first three open reading frames was observed under all three diazotrophic conditions; expression of orf5 was never observed. Genes nifB and fdxN were expressed at similar levels. With Mo, expression of nifO and nifQ was approximately 20- and approximately 400-fold lower than that of fdxN, respectively. Without Mo, expression of nifB dropped three- to fourfold and that of nifQ dropped to the detection limit. However, expression of nifO increased threefold. The products of nifB, fdxN, nifO, and nifQ have been visualized in A. vinelandii as beta-galactosidase fusion proteins with the expected molecular masses. The NifB- fusion lacked activity for any of the three nitrogenase systems and showed an iron-molybdenum cofactor-deficient phenotype in the presence of Mo. The FdxN- mutation resulted in reduced nitrogenase activities, especially when V was present. Dinitrogenase activity in extracts was similarly affected, suggesting a role of FdxN in iron-molybdenum cofactor synthesis. The NifO(-)-producing mutation did not affect any of the nitrogenases under standard diazotrophic conditions. The NifQ(-)-producing mutation resulted in an increased (approximately 1,000-fold) Mo requirement for Mo nitrogenase activity, a phenotype already observed with Klebsiella pneumoniae. No effect of the NifQ(-)-producing mutation on V or Fe nitrogenase was found; this is consistent with its very low expression under those conditions. Mutations in orf5 had no effect on nitrogenase activity. Images PMID:8491713

  11. 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.

  12. Alkyne Substrate Interaction within the Nitrogenase MoFe Protein¶

    PubMed Central

    Dos Santos, Patricia C.; Mayer, Suzanne M.; Barney, Brett M.; Seefeldt, Lance C.; Dean, Dennis R.

    2009-01-01

    Nitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC≡CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein α-70Val residue, whose side-chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of α-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C-CH3), whereas substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the α-70Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C-CH2-CH3). This protein shows no detectable reduction of the internal alkyne 2-butyne (H3C-C≡C-CH3). In contrast, substitution of the nearby α-191Gln residue by alanine, in combination with the α-70Ala substitution, does result in significant reduction 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein. PMID:17610955

  13. Nitrogenase in the archaebacterium Methanosarcina barkeri 227.

    PubMed Central

    Lobo, A L; Zinder, S H

    1990-01-01

    The discovery of nitrogen fixation in the archaebacterium Methanosarcina barkeri 227 raises questions concerning the similarity of archaebacterial nitrogenases to Mo and alternative nitrogenases in eubacteria. A scheme for achieving a 20- to 40-fold partial purification of nitrogenase components from strain 227 was developed by using protamine sulfate precipitation, followed by using a fast protein liquid chromatography apparatus operated inside an anaerobic glove box. As in eubacteria, the nitrogenase activity was resolved into two components. The component 1 analog had a molecular size of approximately 250 kDa, as estimated by gel filtration, and sodium dodecyl sulfate-polyacrylamide gels revealed two predominant bands with molecular sizes near 57 and 62 kDa, consistent with an alpha 2 beta 2 tetramer as in eubacterial component 1 proteins. For the component 2 analog, a molecular size of approximately 120 kDa was estimated by gel filtration, with a subunit molecular size near 31 kDa, indicating that the component 2 protein is a tetramer, in contrast to eubacterial component 2 proteins, which are dimers. Rates of C2H2 reduction by the nearly pure subunits were 1,000 nmol h-1 mg of protein-1, considerably lower than those for conventional Mo nitrogenases but similar to that of the non-Mo non-V nitrogenase from Azotobacter vinelandii. Strain 227 nitrogenase reduced N2 at a higher rate per electron than it reduced C2H2, also resembling the non-Mo non-V nitrogenase of A. vinelandii. Ethane was not produced from C2H2. NH4+ concentrations as low as 10 microM caused a transient inhibition of C2H2 reduction by strain 227 cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2254255

  14. Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus.

    PubMed

    Siemann, Stefan; Schneider, Klaus; Oley, Mareke; Müller, Achim

    2003-04-01

    In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (1 microM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (1 mM), was capable of partially (approximately 25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of 1 W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N(2) and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H(2) from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E(m) < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E(m) approximately -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20

  15. Climbing Nitrogenase: Towards a Mechanism of Enzymatic Nitrogen Fixation

    PubMed Central

    Dean, Dennis R.; Seefeldt, Lance C.

    2009-01-01

    Conspectus “Nitrogen fixation”—the reduction of dinitrogen (N2) to two ammonia (NH3) molecules—by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase make it the ‘Everest of enzymes’. This Account describes our efforts to climb one “face” of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMocofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, we need to discriminate between the two classes of potential reaction pathways: 1) a “distal” (D) pathway, in which H atoms add sequentially at a single N or 2) an “alternating” (A) pathway, in which H atoms add alternately to the two N atoms of N2. Secondly, we need to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires us to experimentally determine both the number of electrons/protons delivered to the MoFe protein and their “inventory”—a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this “climb” has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-cofactor as the specific site of reactivity, indicated that the sidechain of residue α70V controls access to this face, and supported the involvement of the sidechain of residue α195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use ENDOR

  16. 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.

  17. Identification of an alternative nitrogenase system in Rhodospirillum rubrum.

    PubMed Central

    Lehman, L J; Roberts, G P

    1991-01-01

    A second nitrogenase activity has been demonstrated in Rhodospirillum rubrum. This nitrogenase is expressed whenever a strain lacks an active Mo nitrogenase because of physiological or genetic inactivation. The alternative nitrogenase is able to support growth on N2 in the absence of fixed N. V does not stimulate, nor does Mo or W inhibit, growth or activity under the conditions tested. The proteins responsible for this activity were identified by electrophoretic and immunological properties. The synthesis of these proteins was repressed by NH4+. The alternative nitrogenase reductase is ADP ribosylated in response to darkness by the system that regulates the activity of the Mo nitrogenase. The genes for the alternative nitrogenase have been cloned, and the alternative nitrogenase reductase has been expressed in an in vitro transcription-translation system. Images PMID:1909322

  18. Cyanamide: a new substrate for nitrogenase.

    PubMed

    Miller, R W; Eady, R R

    1988-02-10

    (1) Cyanamide (N identical to C-NH2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent Km values near 0.8 mM. (2) Reduction products were CH4, CH3NH2 and NH3 formed by pathways requiring 6 or 8 electrons: N identical to CNH2 + 6e + 6H+----CH3NH2 + NH3; N identical to CNH2 + 8e + 8H+----CH4 + 2NH3 (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent Km of 2.6 mM showed weaker binding and an 8-fold lower Vmax than did either Mo-nitrogenase. (5) The component ratios of nitrogenase proteins favouring CH4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.

  19. Metal substitution in the active site of nitrogenase MFe(7)S(9) (M = Mo(4+), V(3+), Fe(3+)).

    PubMed

    Lovell, Timothy; Torres, Rhonda A; Han, Wen-Ge; Liu, Tiqing; Case, David A; Noodleman, Louis

    2002-11-01

    The unifying view that molybdenum is the essential component in nitrogenase has changed over the past few years with the discovery of a vanadium-containing nitrogenase and an iron-only nitrogenase. The principal question that has arisen for the alternative nitrogenases concerns the structures of their corresponding cofactors and their metal-ion valence assignments and whether there are significant differences with that of the more widely known molybdenum-iron cofactor (FeMoco). Spin-polarized broken-symmetry (BS) density functional theory (DFT) calculations are used to assess which of the two possible metal-ion valence assignments (4Fe(2+)4Fe(3+) or 6Fe(2+)2Fe(3+)) for the iron-only cofactor (FeFeco) best represents the resting state. For the 6Fe(2+)2Fe(3+) oxidation state, the spin coupling pattern for several spin state alignments compatible with S = 0 were generated and assessed by energy criteria. The most likely BS spin state is composed of a 4Fe cluster with spin S(a) = (7)/(2) antiferromagnetically coupled to a 4Fe' cluster with spin S(b) = (7)/(2). This state has the lowest DFT energy for the isolated FeFeco cluster and displays calculated Mössbauer isomer shifts consistent with experiment. Although the S = 0 resting state of FeFeco has recently been proposed to have metal-ion valencies of 4Fe(2+)4Fe(3+) (derived from experimental Mössbauer isomer shifts), our isomer shift calculations for the 4Fe(2+)4Fe(3+) oxidation state are in poorer agreement with experiment. Using the Mo(4+)6Fe(2+)Fe(3+) oxidation level of the cofactor as a starting point, the structural consequences of replacement of molybdenum (Mo(4+)) with vanadium (V(3+)) or iron (Fe(3+)) in the cofactor have been investigated. The size of the cofactor cluster shows a dependency on the nature of the heterometal and increases in the order FeMoco < FeVco < FeFeco.

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

    PubMed

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

    2016-08-23

    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 NifEN(M)) 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 NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) 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, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αβ-dimer of NifEN(M) 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

  1. Molybdenum cofactor deficiency.

    PubMed

    Atwal, Paldeep S; Scaglia, Fernando

    2016-01-01

    Molybdenum cofactor deficiency (MoCD) is a severe autosomal recessive inborn error of metabolism first described in 1978. It is characterized by a neonatal presentation of intractable seizures, feeding difficulties, severe developmental delay, microcephaly with brain atrophy and coarse facial features. MoCD results in deficiency of the molybdenum cofactor dependent enzymes sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase and mitochondrial amidoxime reducing component. The resultant accumulation of sulfite, taurine, S-sulfocysteine and thiosulfate contributes to the severe neurological impairment. Recently, initial evidence has demonstrated early treatment with cyclic PMP can turn MoCD type A from a previously neonatal lethal condition with only palliative options, to near normal neurological outcomes in affected patients. We review MoCD and focus on describing the currently published evidence of this exciting new therapeutic option for MoCD type A caused by pathogenic variants in MOCD1.

  2. 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.

  3. 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).

  4. Structural Framework for Metal Incorporation during Molybdenum Cofactor Biosynthesis.

    PubMed

    Kasaragod, Vikram Babu; Schindelin, Hermann

    2016-05-01

    The molybdenum cofactor (Moco) is essential for the catalytic activity of all molybdenum-containing enzymes with the exception of nitrogenase. Moco biosynthesis follows an evolutionarily highly conserved pathway and genetic deficiencies in the corresponding human enzymes result in Moco deficiency, which manifests itself in severe neurological symptoms and death in childhood. In humans the final steps of Moco biosynthesis are catalyzed by gephyrin, specifically the penultimate adenylation of molybdopterin (MPT) by its N-terminal G domain (GephG) and the final metal incorporation by its C-terminal E domain (GephE). To better understand the poorly defined molecular framework of this final step, we determined high-resolution crystal structures of GephE in the apo state and in complex with ADP, AMP, and molybdate. Our data provide novel insights into the catalytic steps leading to final Moco maturation, namely deadenylation as well as molybdate binding and insertion. PMID:27112598

  5. Kinetic studies of Bacillus polymyxa nitrogenase.

    PubMed Central

    Hermann, T E; Wilson, P W

    1976-01-01

    Nitrogenase from the facultative anaerobe Bacillus polymxa was separated into its component proteins, which were recombined in the ratio that produced optimal specific activity (125 to 175 nmol of C2H2 reduced/min per mg of total protein). The apparent Michaelis constants (Km)for the magnesium adenosine triphosphate complex, reducible substrates azide, acetylene, and N2 and the nonphysiological electron donor hydrosulfite (S2O42-) were determined to be 0.7, 0.7, 0.2, 0.06, and 0.03 MM, respectively. These apparent Km values are in reasonable agreement with those reported for the nitrogenases of Azotobacter vinelandii and Klebsiella pneumoniae. Either a total lack of cooperativity between binding sites or a single binding site for reducible substrates is indicated by analysis of Hill plots. Hill plot slopes of approximately 1.7 suggest that multiple binding sites exist for both ATP and S2O42-. PMID:770451

  6. 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

  7. Isolation of Nitrogen-Fixing Bacteria Containing Molybdenum-Independent Nitrogenases from Natural Environments

    PubMed Central

    Loveless, Telisa M.; Saah, J. Royden; Bishop, Paul E.

    1999-01-01

    Seven diazotrophs that grow well under Mo-deficient, N2-fixing conditions were isolated from a variety of environments. These isolates fall in the γ subdivision of the class Proteobacteria and have genes that encode the Mo nitrogenase (nitrogenase 1) and the V nitrogenase (nitrogenase 2). Four of the isolates also harbor genes that encode the iron-only nitrogenase (nitrogenase 3). PMID:10473439

  8. Cofactor squelching: Artifact or fact?

    PubMed

    Schmidt, Søren Fisker; Larsen, Bjørk Ditlev; Loft, Anne; Mandrup, Susanne

    2016-07-01

    Cofactor squelching is the term used to describe competition between transcription factors (TFs) for a limited amount of cofactors in a cell with the functional consequence that TFs in a given cell interfere with the activity of each other. Since cofactor squelching was proposed based primarily on reporter assays some 30 years ago, it has remained controversial, and the idea that it could be a physiologically relevant mechanism for transcriptional repression has not received much support. However, recent genome-wide studies have demonstrated that signal-dependent TFs are very often absent from the enhancers that are acutely repressed by those signals, which is consistent with an indirect mechanism of repression such as squelching. Here we review these recent studies in the light of the classical studies of cofactor squelching, and we discuss how TF cooperativity in so-called hotspots and super-enhancers may sensitize these to cofactor squelching.

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

    PubMed

    Dance, Ian

    2012-07-01

    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.

  10. Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes

    PubMed Central

    2012-01-01

    Background The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Results Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. Conclusions Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase

  11. 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.

  12. 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.

  13. 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.

    2016-07-01

    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.

  14. ATP-independent substrate reduction by nitrogenase P-cluster variant

    PubMed Central

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

    2012-01-01

    The P-cluster of nitrogenase is largely known for its function to mediate electron transfer to the active cofactor site during catalysis. Here, we show that a P-cluster variant (designated P*-cluster), which consists of paired [Fe4S4]-like clusters, can catalyze ATP-independent substrate reduction in the presence of a strong reductant, europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA]. The observation of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ, and ΔnifB MoFe protein, which corresponds to a decrease of the amount of P*-clusters in these cofactor-deficient proteins, firmly establishes P*-cluster as a catalytically active metal center in Eu(II)-DTPA–driven reactions. More excitingly, the fact that P*-cluster is not only capable of catalyzing the two-electron reduction of proton, acetylene, ethylene, and hydrazine, but also capable of reducing cyanide, carbon monoxide, and carbon dioxide to alkanes and alkenes, points to a possibility of developing biomimetic catalysts for hydrocarbon production under ambient conditions. PMID:22509042

  15. Tyrosine-Coordinated P-Cluster in G. diazotrophicus Nitrogenase: Evidence for the Importance of O-Based Ligands in Conformationally Gated Electron Transfer.

    PubMed

    Owens, Cedric P; Katz, Faith E H; Carter, Cole H; Oswald, Victoria F; Tezcan, F Akif

    2016-08-17

    The P-cluster is a unique iron-sulfur center that likely functions as a dynamic electron (e(-)) relay site between the Fe-protein and the catalytic FeMo-cofactor in nitrogenase. The P-cluster has been shown to undergo large conformational changes upon 2-e(-) oxidation which entail the coordination of two of the Fe centers to a Ser side chain and a backbone amide N, respectively. Yet, how and if this 2-e(-) oxidized state (P(OX)) is involved in catalysis by nitrogenase is not well established. Here, we present the crystal structures of reduced and oxidized MoFe-protein (MoFeP) from Gluconacetobacter diazotrophicus (Gd), which natively possesses an Ala residue in the position of the Ser ligand to the P-cluster. While reduced Gd-MoFeP is structurally identical to previously characterized counterparts around the FeMo-cofactor, oxidized Gd-MoFeP features an unusual Tyr coordination to its P-cluster along with ligation by a backbone amide nitrogen. EPR analysis of the oxidized Gd-MoFeP P-cluster confirmed that it is a 2-e(-) oxidized, integer-spin species. Importantly, we have found that the sequence positions corresponding to the Ser and Tyr ligands are almost completely covariant among Group I nitrogenases. These findings strongly support the possibility that the P(OX) state is functionally relevant in nitrogenase catalysis and that a hard, O-based anionic ligand serves to stabilize this state in a switchable fashion. PMID:27487256

  16. 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.

  17. Genetics Home Reference: molybdenum cofactor deficiency

    MedlinePlus

    ... molybdenum, is essential to the function of several enzymes. These enzymes help break down (metabolize) different substances in the ... molybdenum cofactor biosynthesis. Without the cofactor, the metabolic enzymes that rely on it cannot function. The resulting ...

  18. Functional diversity of organic molecule enzyme cofactors.

    PubMed

    Richter, Michael

    2013-10-11

    Covering up to 2013. Cofactor-dependent enzymes which need small organic molecule cofactors to accomplish enzymatic activity are widespread. The number of newly discovered reactions within cofactor-dependent enzyme families increases continuously. A knowledge based categorization of cofactors with respect to their chemical properties shows that within enzymes they are not used for the catalysis of singular types of reactions and functionalities. Therefore, in many cases an unambiguous and narrow classification does not seem sufficient. Rather, the functional diversity of cofactors found in enzymes represents variations around specific themes with regard to a catalysed reaction and the cofactor chemistry. Furthermore, organic cofactor molecules are also used for non-enzymatic functions. Two representative cofactors are exemplarily discussed in detail, thiamin diphosphate (ThDP) as a self-sufficient cofactor and S-adenosyl-L-methionine (SAM), as both a catalytic and building-block-delivering cofactor. A further synopsis on selected examples of organic cofactors emphasizes the discovery and application of new enzymatic activities based on the cofactor-dependent chemistry and shows how bioinspired synthesis approaches expand catalytic and non-catalytic synthesis applications beyond natural solutions.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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

  1. 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.

  2. 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.

  3. Organization of potential alternative nitrogenase genes from Clostridium pasteurianum.

    PubMed

    Zinoni, F; Robson, R M; Robson, R L

    1993-07-18

    A 3.3 kb HindIII genomic DNA fragment from Clostridium pasteurianum ATCC 6013 which hybridized to the anfDGK genes for the Fe-only 'alternative' nitrogenase from Azotobacter vinelandii was cloned. Open reading frames (ORFs D, G, and K) with high sequence identity to anfD, anfG, and part of anfK were located in the nucleotide sequence obtained for 2494 bp of this fragment. In C. pasteurianum, ORFD maps approximately 1.8 kb downstream of nifH3 and is transcribed in the same direction. There was no evidence for additional copies of ORFDGK-like sequences in the genome of C. pasteurianum, other than those encoding the Mo-nitrogenase. Physiological and biochemical studies suggest that a nitrogenase not requiring molybdenum may occur in C. pasteurianum. This enzyme is probably encoded by nifH3 and ORFs D, G, and K identified here. PMID:8334167

  4. Effect of Rice Plants on Nitrogenase Activity of Flooded Soils

    PubMed Central

    Habte, Mitiku; Alexander, Martin

    1980-01-01

    In samples of flooded soil containing blue-green algae (cyanobacteria), the presence of rice plants did not influence the nitrogenase activity of the algae. Nitrogenase activity of heterotrophic bacteria was enhanced by the presence of rice plants, but this activity was not affected by changes in plant density. The rate of nitrogen fixation in the rhizosphere, however, varied significantly among the 16 rice varieties tested. A simple method was devised to test the nitrogen-fixing activity in the root zone of rice varieties, and data were obtained showing marked differences in the activities of the 16 varieties. In tests of two varieties with dissimilar rates of nitrogen fixation in their rhizospheres, the variety which had the greater root weight and lesser shoot weight and which supported greater methane formation had the greater nitrogenase activity. PMID:16345630

  5. Effect of rice plants on nitrogenase activity of flooded soils.

    PubMed

    Habte, M; Alexander, M

    1980-09-01

    In samples of flooded soil containing blue-green algae (cyanobacteria), the presence of rice plants did not influence the nitrogenase activity of the algae. Nitrogenase activity of heterotrophic bacteria was enhanced by the presence of rice plants, but this activity was not affected by changes in plant density. The rate of nitrogen fixation in the rhizosphere, however, varied significantly among the 16 rice varieties tested. A simple method was devised to test the nitrogen-fixing activity in the root zone of rice varieties, and data were obtained showing marked differences in the activities of the 16 varieties. In tests of two varieties with dissimilar rates of nitrogen fixation in their rhizospheres, the variety which had the greater root weight and lesser shoot weight and which supported greater methane formation had the greater nitrogenase activity. PMID:16345630

  6. Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

    PubMed Central

    May, H D; Schauer, N L; Ferry, J G

    1986-01-01

    The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant. PMID:3700335

  7. Isotope fractionation by alternative nitrogenases and past ocean anoxia

    NASA Astrophysics Data System (ADS)

    Zhang, X.; Sigman, D. M.; Kraepiel, A. M.

    2013-12-01

    The budget of fixed nitrogen (N) in the ocean, a key limiting nutrient for marine ecosystems, is dominated by N2 fixation as the input and denitrification as the output. The 15N/14N of marine N (quantified by δ15N) is believed to be set by two parameters: (1) the δ15N of newly fixed N and (2) the net isotopic fractionation associated with denitrification, which elevates the δ15N of marine N above that of newly fixed N. If so, the δ15N of oceanic fixed N cannot drop below the δ15N of the N produced by N2 fixation, currently believed to be -2 to 0‰. Yet significantly lower δ15N has been measured in sedimentary organic matter from the past, for example, the Archean Eon (-6‰) and mid-Cretaceous oceanic anoxic events (OAEs, -5‰). Here we show that the δ15N of newly fixed N can be as low as -7‰, depending on the type of nitrogenase that catalyzes N2 fixation. Vanadium (V)- or iron (Fe)- only based 'alternative' nitrogenases produce fixed N that is significantly lower in 15N than the more common Mo-based nitrogenase (-6‰ and -7‰ for V- and Fe-nitrogenase, respectively, versus -2‰ for Mo-nitrogenase), regardless of N2-fixer phylogeny or metabolism. Consistent with a Mo-poor Archean ocean and the preferential scavenging of seawater Mo compared to V and Fe into low oxygen/sulfidic OAE sediments, a N cycle in which alternative nitrogenases accounted for a large fraction of N2 fixation helps explain the low sedimentary δ15N from these periods. Our results imply that the role of alternative nitrogenases may have been important in low oxygen environments of the past, suggesting that they are also important in modern low oxygen settings. Elucidating the conditions under which alternative nitrogenases contribute to N2 fixation is necessary to understanding the evolution of the oceanic N budget through time.

  8. 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.

  9. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:27630531

  11. The nitrogenase proteins of Rhizobium meliloti: purification and properties of the MoFe and Fe components.

    PubMed

    Miller, R W; Yu, Z; Zarkadas, C G

    1993-04-21

    The alfalfa-Rhizobium meliloti symbiosis contributes a major portion of biologically fixed nitrogen to temperate zone forage crop production. Highly-purified molybdenum-iron (MoFe) and iron (Fe) nitrogenase components were obtained for the first time from extracts of R. meliloti bacteroids. Intact bacteroid cells were isolated anaerobically from 100 g quantities of alfalfa nodules following storage in liquid nitrogen. Centrifuged bacteroid extracts showed a marked reduction in specific activity when assayed at protein concentrations less than 1 mg/ml. Both nitrogenase proteins were resolved and purified to homogeneity as determined spectroscopically and by SDS-PAGE. The purified MoFe protein differed in several respects from previously characterized nitrogenase proteins. Saturation of the acetylene-reducing and proton-reducing activities of the R. meliloti MoFe protein required higher relative concentrations of Fe protein than nitrogenase proteins purified from free living diazotrophs. Electron allocation to dinitrogen reduction was sustained at component ratios similar to those present in bacteroid extracts, suggesting that while the observed saturation effects were not detrimental to physiological function in the symbiotic system, overall activity could be enhanced by higher levels of iron protein. Analyses of the MoFe protein gave 22 Fe, 22 labile sulfide and 1.7 Mo atoms per molecular unit of 215 kDa. Dithionite-reduced MoFe protein contained a spin 3/2 iron centre but had a lower visible absorbance at 360 nm than the equivalent Azotobacter chroococcum component. Amino-acid composition indicated a notably lesser tryptophan content, and cysteine content greater than that of the equivalent tetrameric protein of free living diazotrophs. Ratios of acidic and basic residues were similar to other MoFe proteins. Calculation of hydrophobicity and discriminant parameters gave values midway between those expected for soluble cytoplasmic proteins and peripheral membrane

  12. 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

  13. 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…

  14. Properties of a reaction-bonded β-SiAlON ceramic doped with an FeMo alloy for application to molten aluminum environments

    NASA Astrophysics Data System (ADS)

    Li, Yan-jun; Yu, Hai-liang; Jin, Hai-yun; Shi, Zhong-qi; Qiao, Guan-jun; Jin, Zhi-hao

    2015-05-01

    An FeMo-alloy-doped β-SiAlON (FeMo/β-SiAlON) composite was fabricated via a reaction-bonding method using raw materials of Si, Al2O3, AlN, FeMo, and Sm2O3. The effects of FeMo on the microstructure and mechanical properties of the composite were investigated. Some properties of the composite, including its bending strength at 700°C and after oxidization at 700°C for 24 h in air, thermal shock resistance and corrosion resistance to molten aluminum, were also evaluated. The results show that the density, toughness, bending strength, and thermal shock resistance of the composite are obviously improved with the addition of an FeMo alloy. In addition, other properties of the composite such as its high-temperature strength and oxidized strength are also improved by the addition of FeMo alloy, and its corrosion resistance to molten aluminum is maintained. These findings indicate that the developed FeMo/β-SiAlON composite exhibits strong potential for application to molten aluminum environments.

  15. 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.

  16. [Hpv cofactors in cervical carcinogenesis].

    PubMed

    Pinto, Alvaro P; Tulio, Siumara; Cruz, Olívia Russo

    2002-01-01

    Human papillomavirus (HPV) plays a central rule in uterine cervix carcinogenesis. Other factors direct or indirectly influence the installation of this mechanism in cervical squamous epithelium. Investigations regarding mechanisms of interaction of these factors with viral elements are found in the literature of the last 20 years. The present review article discusses possible co-factors of HPV in the genesis of the squamous carcinoma of uterine cervix, taking into account only the factors whose association with the virus or cervical cancer has been documented by experimental studies, and not based just on clinical or epidemiological data. Among the approached parameters are immunological factors (local and humoral immune response), the association with Acquired Immune Deficiency Syndrome, genetic factors as protein p53 polymorphism, tabagism and the use of oral contraceptives. All these factors interact in variable intensity with oncoproteins and other HPV elements, increasing and facilitating the virus action in host cells, leading to the development of immortalization and carcinogenesis. PMID:12185639

  17. 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

  18. Characterization of molybdenum cofactor from Escherichia coli.

    PubMed Central

    Amy, N K; Rajagopalan, K V

    1979-01-01

    Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity. PMID:387715

  19. An Evolved Orthogonal Enzyme/Cofactor Pair.

    PubMed

    Reynolds, Evan W; McHenry, Matthew W; Cannac, Fabien; Gober, Joshua G; Snow, Christopher D; Brustad, Eric M

    2016-09-28

    We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.

  20. Assay of nitrogenase activity in intact plant systems.

    PubMed

    Jain, M K; Vlassak, K

    1975-01-01

    Nitrogenase activity was assayed in intact system of Cichorium intybus, a non-leguminous commercially cultivated crop, Dahlia pinnata and Helianthus annus, and Taraxacum officinale, a common weed plant. The assay was made in fabricated cylinders which could accomodate pot with plants. In such kind of assay along with rhizosphere microflora, the nitrogen fixed by phyllosphere nitrogen fixing microflora could also be accounted, which otherwise was difficult to be accounted for. PMID:1211718

  1. 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.)

  2. Identification of a key catalytic intermediate demonstrates that nitrogenase is activated by the reversible exchange of N₂ for H₂.

    PubMed

    Lukoyanov, Dmitriy; Yang, Zhi-Yong; Khadka, Nimesh; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

    2015-03-18

    Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3. PMID:25741750

  3. IR-Monitored Photolysis of CO-Inhibited Nitrogenase: A Major EPR-Silent Species with Coupled Terminal CO Ligands

    PubMed Central

    Yan, Lifen; Dapper, Christie H.; Scott, Aubrey D.; Newton, William E.

    2015-01-01

    We have used Fourier transform infrared spectroscopy (FT-IR) to observe the photolysis and recombination of a novel EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K yields a strong negative IR difference band at 1938 cm−1, along with a weaker negative feature at 1911 cm−1. These bands and the associated chemical species have both been assigned the label ‘Hi-3’. A positive band at 1921 cm−1 is assigned to the ‘Lo-3’ photoproduct. By using an isotopic mixture of 12C16O and 13C18O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at ~1917 cm−1. Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200–240 K we found that recombination to Hi-3 does indeed occur, with an activation energy of ~6.5 kJ mol−1. The frequencies of the Hi-3 bands suggest terminal CO ligation. We tested this hypothesis with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S = 0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at 1938 and 1909 cm−1 respectively, with relative band intensities of ~3.5:1, in good agreement with experiment. From the observed IR intensities, we find that Hi-3 is present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered Fischer-Tropsch chemistry is discussed. PMID:23136072

  4. Co-factor activated recombinant adenovirus proteinases

    SciTech Connect

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

    PubMed Central

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

    2015-01-01

    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 (106 and 1012 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 106 cfu/ml inoculum, whereas the higher inoculum size (1012 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. PMID:26868594

  7. Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

    PubMed

    Loveless, T M; Bishop, P E

    1999-04-01

    A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase. PMID:10420583

  8. Analysis of Genes Encoding an Alternative Nitrogenase in the Archaeon Methanosarcina barkeri 227

    PubMed Central

    Chien, Yueh-Tyng; Auerbuch, Victoria; Brabban, Andrew D.; Zinder, Stephen H.

    2000-01-01

    Methanosarcina barkeri 227 possesses two clusters of genes potentially encoding nitrogenases. We have previously demonstrated that one cluster, called nif2, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium Clostridium pasteurianum. The previously cloned nifH1 from M. barkeri shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases. In this study, we cloned and sequenced nifD1 and part of nifK1 from M. barkeri 227. The deduced amino acid sequence encoded by nifD1 from M. barkeri showed great similarity with vnfD gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the vnfD gene product from Anabaena variabilis. Moreover, there was a small open reading frame located between nifD1 and nifK1 with clear homology to vnfG, a hallmark of eubacterial alternative nitrogenases. Stimulation of diazotrophic growth of M. barkeri 227 by V in the absence of Mo was demonstrated. The unusual complement of nif genes in M. barkeri 227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes. PMID:10809706

  9. Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

    PubMed

    Loveless, T M; Bishop, P E

    1999-04-01

    A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

  10. Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

    PubMed Central

    Lobo, Anthony L.; Zinder, Stephen H.

    1988-01-01

    Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N2-grown cultures than in NH4+-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h−1 mg of protein−1), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C2H4 formed h−1 mg of protein−1 when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C2H2

  11. Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase

    SciTech Connect

    Parsons, R.; Silvester, W.B.; Harris, S. ); Gruijters, W.T.M.; Bullivant, S. )

    1987-01-01

    When Frankia HFPCcI3 was grown in culture at oxygen O{sub 2} levels ranging from 2 to 70 kilopascals O{sub 2}, under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO{sub 2} and showed a marked optimum close to growth pO{sub 2}. Vesicles were thin walled at low pO{sub 2} and very thick walled at high pO{sub 2}. Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO{sub 2}.

  12. Cofactor modification analysis: a computational framework to identify cofactor specificity engineering targets for strain improvement.

    PubMed

    Lakshmanan, Meiyappan; Chung, Bevan Kai-Sheng; Liu, Chengcheng; Kim, Seon-Won; Lee, Dong-Yup

    2013-12-01

    Cofactors, such as NAD(H) and NADP(H), play important roles in energy transfer within the cells by providing the necessary redox carriers for a myriad of metabolic reactions, both anabolic and catabolic. Thus, it is crucial to establish the overall cellular redox balance for achieving the desired cellular physiology. Of several methods to manipulate the intracellular cofactor regeneration rates, altering the cofactor specificity of a particular enzyme is a promising one. However, the identification of relevant enzyme targets for such cofactor specificity engineering (CSE) is often very difficult and labor intensive. Therefore, it is necessary to develop more systematic approaches to find the cofactor engineering targets for strain improvement. Presented herein is a novel mathematical framework, cofactor modification analysis (CMA), developed based on the well-established constraints-based flux analysis, for the systematic identification of suitable CSE targets while exploring the global metabolic effects. The CMA algorithm was applied to E. coli using its genome-scale metabolic model, iJO1366, thereby identifying the growth-coupled cofactor engineering targets for overproducing four of its native products: acetate, formate, ethanol, and lactate, and three non-native products: 1-butanol, 1,4-butanediol, and 1,3-propanediol. Notably, among several target candidates for cofactor engineering, glyceraldehyde-3-phosphate dehydrogenase (GAPD) is the most promising enzyme; its cofactor modification enhanced both the desired product and biomass yields significantly. Finally, given the identified target, we further discussed potential mutational strategies for modifying cofactor specificity of GAPD in E. coli as suggested by in silico protein docking experiments.

  13. Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

    PubMed

    Maynard, R H; Premakumar, R; Bishop, P E

    1994-09-01

    Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.

  14. 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.

  15. 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

  16. Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii.

    PubMed

    Joerger, R D; Jacobson, M R; Bishop, P E

    1989-06-01

    Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo- and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the Tn5 insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A + T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-1 and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Mo- and V-deficient conditions.

  17. 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.

  18. 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.

  19. Activation of Inactive Nitrogenase by Acid-Treated Component I

    PubMed Central

    Nagatani, H. H.; Shah, Vinod K.; Brill, Winston J.

    1974-01-01

    When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N2. Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N2-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum. PMID:4218230

  20. 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.

  1. 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.

  2. 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

  3. 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. PMID:26343576

  4. 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.

  5. High-Performance Anode Material Sr2FeMo0.65Ni0.35O6-δ with In Situ Exsolved Nanoparticle Catalyst.

    PubMed

    Du, Zhihong; Zhao, Hailei; Yi, Sha; Xia, Qing; Gong, Yue; Zhang, Yang; Cheng, Xing; Li, Yan; Gu, Lin; Świerczek, Konrad

    2016-09-27

    A metallic nanoparticle-decorated ceramic anode was prepared by in situ reduction of the perovskite Sr2FeMo0.65Ni0.35O6-δ (SFMNi) in H2 at 850 °C. The reduction converts the pure perovksite phase into mixed phases containing the Ruddlesden-Popper structure Sr3FeMoO7-δ, perovskite Sr(FeMo)O3-δ, and the FeNi3 bimetallic alloy nanoparticle catalyst. The electrochemical performance of the SFMNi ceramic anode is greatly enhanced by the in situ exsolved Fe-Ni alloy nanoparticle catalysts that are homogeneously distributed on the ceramic backbone surface. The maximum power densities of the La0.8Sr0.2Ga0.8Mg0.2O3-δ electrolyte supported a single cell with SFMNi as the anode reached 590, 793, and 960 mW cm(-2) in wet H2 at 750, 800, and 850 °C, respectively. The Sr2FeMo0.65Ni0.35O6-δ anode also shows excellent structural stability and good coking resistance in wet CH4. The prepared SFMNi material is a promising high-performance anode for solid oxide fuel cells. PMID:27529355

  6. High-Performance Anode Material Sr2FeMo0.65Ni0.35O6-δ with In Situ Exsolved Nanoparticle Catalyst.

    PubMed

    Du, Zhihong; Zhao, Hailei; Yi, Sha; Xia, Qing; Gong, Yue; Zhang, Yang; Cheng, Xing; Li, Yan; Gu, Lin; Świerczek, Konrad

    2016-09-27

    A metallic nanoparticle-decorated ceramic anode was prepared by in situ reduction of the perovskite Sr2FeMo0.65Ni0.35O6-δ (SFMNi) in H2 at 850 °C. The reduction converts the pure perovksite phase into mixed phases containing the Ruddlesden-Popper structure Sr3FeMoO7-δ, perovskite Sr(FeMo)O3-δ, and the FeNi3 bimetallic alloy nanoparticle catalyst. The electrochemical performance of the SFMNi ceramic anode is greatly enhanced by the in situ exsolved Fe-Ni alloy nanoparticle catalysts that are homogeneously distributed on the ceramic backbone surface. The maximum power densities of the La0.8Sr0.2Ga0.8Mg0.2O3-δ electrolyte supported a single cell with SFMNi as the anode reached 590, 793, and 960 mW cm(-2) in wet H2 at 750, 800, and 850 °C, respectively. The Sr2FeMo0.65Ni0.35O6-δ anode also shows excellent structural stability and good coking resistance in wet CH4. The prepared SFMNi material is a promising high-performance anode for solid oxide fuel cells.

  7. Control of ubiquitin conjugation by cdc48 and its cofactors.

    PubMed

    Buchberger, Alexander

    2010-01-01

    Cdc48 (alias p97, VCP) is an important motor and regulator for the turnover of ubiquitylated proteins, both in proteasomal degradation and in nonproteolytic pathways. The diverse cellular tasks of Cdc48 are controlled by a large number of cofactors. Substrate-recruiting cofactors mediate the specific recognition of ubiquitylated target proteins, whereas substrate-processing cofactors often exhibit ubiquitin ligase or deubiquitylating activities that enable them to modulate the ubiquitylation state of substrates. This chapter introduces the major groups of Cdc48 cofactors and discusses the versatile options of substrate-processing cofactors to control the fate of Cdc48 substrates.

  8. 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.

  9. Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea.

    PubMed

    Johnsen, Ulrike; Schönheit, Peter

    2007-09-01

    Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn(2+) and exhibited high thermostability up to 70 degrees C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.

  10. NanoSIMS Analyses of Mo Indicate Nitrogenase Activity and Help Solve a N and C Fixation Puzzle in a Marine Cyanobacterium

    NASA Astrophysics Data System (ADS)

    Pett-Ridge, J.; Weber, P. K.; Finzi, J.; Hutcheon, I. D.; Capone, D. G.

    2006-12-01

    Diazotrophic cyanobacteria are capable of both CO2 and N2 fixation, yet must separate these two functions because the nitrogenase enzymes used in N2 fixation are strongly inhibited by O2 produced during photosynthesis. Some lineages, such as Anabaena, use specialized cells (heterocysts) to maintain functional segregation. However the mechanism of this segregation is poorly understood in Trichodesmium, a critical component of marine primary production in the tropical and subtropical North Atlantic. While some Trichodesmium studies suggest a temporal segregation of the nitrogen and carbon fixing processes, others indicate nitrogen fixation is spatially isolated in differentiated cells called diazocytes. In order to isolate the intracellular location of N fixation in both species, we used a combination of TEM, SEM and NanoSIMS analysis to map the distribution of C, N and Mo (a critical nitrogenase co-factor) isotopes in intact cells. NanoSIMS is a powerful surface analysis tool which combines nanometer-scale imaging resolution with the high sensitivity of mass spectrometry. Using cells grown in a 13CO^2 and 15N2 enriched atmosphere, our analyses indicate that in Anabaena, heterocysts are consistently enriched in Mo, and Mo accumulation suggests active N fixation (as opposed to N storage). In the non- heterocystous Trichodesmium, Mo is concentrated in sub-regions of individual cells, and is not associated with regions of N storage (cyanophycin granules). We suggest that NanoSIMS mapping of metal enzyme co- factors is a unique method of identifying physiological and morphological characteristics within individual bacterial cells. This combination of NanoSIMS analysis and high resolution microscopy allows isotopic analysis to be linked to morphological features and holds great promise for fine-scale studies of bacteria metabolism.

  11. Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations.

    PubMed

    Pereg Gerk, L; Gilchrist, K; Kennedy, I R

    2000-05-01

    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.

  12. 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

  13. 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.

  14. 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

  15. Nitrogenase gene expression in the Chesapeake Bay Estuary.

    PubMed

    Short, Steven M; Zehr, Jonathan P

    2007-06-01

    Like many estuaries, the Chesapeake Bay has pronounced gradients in salinity and nutrients. Previous studies have shown that there is a high diversity of nitrogenase (nifH) genes in the estuary, and that there are specific distributions of individual nifH phylotypes. In contrast to previous work that revealed the remarkable diversity of nifH phylotypes in the Chesapeake estuary, in this study of nifH expression we only detected two phylotypes, and both were phylogenetically related to cyanobacterial nifH genes. One of the phylotypes was closely related to a nifH sequence from the filamentous, heterocystous cyanobacterium Anabaena cylindrica, and was found at the head of the estuary. The other phylotype was found in a sample collected near the mouth of the estuary and was closely related to nifH sequences from Group A unicellular cyanobacteria, which has previously been reported in oceanic waters only. These nifH phylotypes had distinct patterns of expression that were restricted to different regions of the Chesapeake Bay. This study provides the first evidence of nifH expression in the Chesapeake Bay, and suggests that diazotrophic unicellular cyanobacteria have a broader distribution and activity than previously recognized.

  16. The structures and physicochemical properties of organic cofactors in biocatalysis.

    PubMed

    Fischer, Julia D; Holliday, Gemma L; Rahman, Syed A; Thornton, Janet M

    2010-11-12

    Many crucial biochemical reactions in the cell require not only enzymes for catalysis but also organic cofactors or metal ions. Here, we analyse the physicochemical properties, chemical structures and functions of organic cofactors. Based on a thorough analysis of the literature complemented by our quantitative characterisation and classification, we found that most of these molecules are constructed from nucleotide and amino-acid-type building blocks, as well as some recurring cofactor-specific chemical scaffolds. We show that, as expected, organic cofactors are on average significantly more polar and slightly larger than other metabolites in the cell, yet they cover the full spectrum of physicochemical properties found in the metabolome. Furthermore, we have identified intrinsic groupings among the cofactors, based on their molecular properties, structures and functions, that represent a new way of considering cofactors. Although some classes of cofactors, as defined by their physicochemical properties, exhibit clear structural communalities, cofactors with similar structures can have diverse functional and physicochemical profiles. Finally, we show that the molecular functions of the cofactors not only may duplicate reactions performed by inorganic metal cofactors and amino acids, the cell's other catalytic tools, but also provide novel chemistries for catalysis.

  17. Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd.

    PubMed

    Rai, A N; Rowell, P; Stewart, W D

    1981-03-01

    The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO2; in the absence of CO2 dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO2. Dark CO2 fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO2 in the dark; in the lichen most dark CO2 fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of (14)CO2 in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO2 in the gas phase. Exogenous NH 4

  18. 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.

  19. Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp.

    PubMed

    Habte, M

    1986-11-01

    Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C(2)H(2) --> C(2)H(4)) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed. PMID:16347211

  20. Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp. †

    PubMed Central

    Habte, Mitiku

    1986-01-01

    Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C2H2 → C2H4) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed. PMID:16347211

  1. Hindered rotation of a cofactor methyl group as a probe for protein-cofactor interaction.

    PubMed

    Brosi, Richard; Illarionov, Boris; Mathes, Tilo; Fischer, Markus; Joshi, Monika; Bacher, Adelbert; Hegemann, Peter; Bittl, Robert; Weber, Stefan; Schleicher, Erik

    2010-07-01

    Exploring protein-cofactor interactions on a molecular level is one of the major challenges in modern biophysics. Based on structural data alone it is rarely possible to identify how subtle interactions between a protein and its cofactor modulate the protein's reactivity. In the case of enzymatic processes in which paramagnetic molecules play a certain role, EPR and related methods such as ENDOR are suitable techniques to unravel such important details. In this contribution, we describe how cryogenic-temperature ENDOR spectroscopy can be applied to various LOV domains, the blue-light sensing domains of phototropin photoreceptors, to gain information on the direct vicinity of the flavin mononucleotide (FMN) cofactor by analyzing the temperature dependence of methyl-group rotation attached to C(8) of the FMN's isoalloxazine ring. More specifically, mutational studies of three amino acids surrounding the methyl group led to the identification of Asn425 as an important amino acid that critically influences the dark-state recovery of Avena sativa LOV2 domains. Consequently, it is possible to probe protein-cofactor interactions on a sub-angstrom level by following the temperature dependencies of hyperfine couplings.

  2. Role of cofactors in metalloprotein folding.

    PubMed

    Wilson, Corey J; Apiyo, David; Wittung-Stafshede, Pernilla

    2004-01-01

    Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro , cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro . To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein-metal interactions.

  3. Texture and cofactor zeros of the neutrino mass matrix

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

    We study Majorana neutrino mass matrices that have two texture zeros, or two cofactor zeros, or one texture zero and one cofactor zero. The two texture/cofactor zero conditions give four constraints, which in conjunction with the five measured oscillation parameters completely determine the nine independent real parameters of the neutrino mass matrix. We also study the implications that future measurements of neutrinoless double beta decay and the Dirac CP phase will have on these cases.

  4. 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

  5. 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.

  6. 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. PMID:27529475

  7. Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system.

    PubMed Central

    Jones, B L; Monty, K J

    1979-01-01

    In whole cells of Rhodopseudomonas sphaeroides, nitrogen fixation, as measured by hydrogen production and acetylene reduction, was totally inhibited by micromolar concentrations of ammonia. This inhibition could not be duplicated by glutamate or glutamine alone. The inhibition by ammonia was abolished by methionine sulfoximine, a glutamine synthetase inhibitor. Inhibition by glutamine was complete in the presence of methionine sulfone, a preferential inhibitor of glutamate synthase, presumably by permitting a rise in the glutamine pool. The results indicated that the level of the glutamine pool controlled the activity of nitrogenase. None of these effects could be duplicated with cell-free nitrogenase, indicating there is probably a mediator which responds to the glutamine pool and inhibits nitrogenase, rather than glutamine itself being a direct inhibitor. PMID:314444

  8. 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.

  9. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis.

  10. 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.

  11. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis. PMID:26503676

  12. Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli.

    PubMed

    Harris, G S; White, T C; Flory, J E; Orme-Johnson, W H

    1990-09-15

    A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K

  13. 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.

  14. 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.

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

    SciTech Connect

    Smith, R.L.; Van Baalen, C. ); Tabita, F.R. Ohio State Univ., Columbus )

    1990-05-01

    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% CO{sub 2} in air was inhibited by exposure to 1% CO{sub 2}-99% O{sub 2} and allowed to recover in the presence of high oxygen tensions. Cultures exposed to hyperbaric levels of oxygen in the presence of 10 mM NH{sub 4}NO{sub 3} 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 NH{sub 4}{sup +} rather than NO{sub 3}{sup {minus}}.

  16. 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

  17. 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.

  18. 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. PMID:27102481

  19. Neutrino mass matrices with two vanishing elements/cofactors

    NASA Astrophysics Data System (ADS)

    Dev, S.; Singh, Lal; Raj, Desh

    2015-08-01

    We study the phenomenological implications of the recent neutrino data for class B of two texture zeros and two vanishing cofactors for Majorana neutrinos in the flavor basis. We find that the classes () of two texture zeros and the classes () of two vanishing cofactors have similar predictions for neutrino oscillation parameters for the same mass hierarchy. Similar predictions for the classes () of two texture zeros and classes () of two vanishing cofactors are expected. However, a preference for a shift in the quadrant of the Dirac-type CP-violating phase () in contrast to the earlier analysis has been predicted for a relatively large value of the reactor neutrino mixing angle () for class B of two texture zeros and two vanishing cofactors for an inverted mass spectrum. No such shift in the quadrant of has been found for the normal mass spectrum.

  20. Enzyme cofactors: Double-edged sword for catalysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Ivaylo

    2013-01-01

    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  1. Mössbauer investigation of the cofactor iron of putidamonooxin.

    PubMed

    Bill, E; Bernhardt, F H; Trautwein, A X; Winkler, H

    1985-02-15

    Mononuclear non-heme cofactor iron of putidamonooxin has been investigated in the binary oxidized 'enzyme X substrate' complex and in the ternary 'enzyme X substrate X NO' complex via Mössbauer spectroscopy. The experimental spectra were analyzed on the basis of the spin-Hamiltonian formalism. The resulting fine and hyperfine structure parameters are compared with literature values of similar compounds. From this comparison we conclude that in the binary complex (reduced and oxidized) the mononuclear non-heme cofactor iron has a coordination number higher than four. Additionally, the cofactor iron shows remarkable spectral similarities with iron in protocatechuate 3,4-dioxygenase, though the catalytic properties of the iron sites in the two proteins are different. The data obtained form the ternary 'enzyme X substrate X NO' complex indicate that the cofactor iron (a) is in the ferric intermediate spin state (S = 3/2) and (b) is pentacoordinated, which means that upon NO binding to the reduced cofactor iron at least one ligand has to be released. Comparing our data with literature values suggests that the cofactor iron in the binary as well as in the ternary NO complex is not directly bound to a sulfur atom, though biochemical arguments seem to indicate the opposite.

  2. 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.

  3. 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.

  4. 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

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

    PubMed

    Farmer, Ryan M; Tabita, F Robert

    2015-11-01

    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.

  6. 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.

  7. Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

    PubMed Central

    Spiller, H; Latorre, C; Hassan, M E; Shanmugam, K T

    1986-01-01

    A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment. PMID:2867990

  8. Inhibition of nitrogenase by oxygen in marine cyanobacteria controls the global nitrogen and oxygen cycles

    NASA Astrophysics Data System (ADS)

    Berman-Frank, I.; Chen, Y.-B.; Gerchman, Y.; Dismukes, G. C.; Falkowski, P. G.

    2005-03-01

    Cyanobacterial N2-fixation supplies the vast majority of biologically accessible inorganic nitrogen to nutrient-poor aquatic ecosystems. The process, catalyzed by the heterodimeric protein complex, nitrogenase, is thought to predate that of oxygenic photosynthesis. Remarkably, while the enzyme plays such a critical role in Earth's biogeochemical cycles, the activity of nitrogenase in cyanobacteria is markedly inhibited in vivo at a post-translational level by the concentration of O2 in the contemporary atmosphere leading to metabolic and biogeochemical inefficiency in N2 fixation. We illustrate this crippling effect with data from Trichodesmium spp. an important contributor of "new nitrogen" to the world's subtropical and tropical oceans. The enzymatic inefficiency of nitrogenase imposes a major elemental taxation on diazotrophic cyanobacteria both in the costs of protein synthesis and for scarce trace elements, such as iron. This restriction has, in turn, led to a global limitation of fixed nitrogen in the contemporary oceans and provides a strong biological control on the upper bound of oxygen concentration in Earth's atmosphere.

  9. Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured Frania

    SciTech Connect

    Silvester, W.B.; Winship, L.J. )

    1990-02-01

    Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S.Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O{sub 2} partial pressure (pO{sub 2}) rise, to the proximity of the starting and ending oxygen tensions to the pO{sub 2} optimum, and to the time for which the plant was exposed to the lower pO{sub 2}. Oxygen-induced transients, induced both by step jumps in pO{sub 2} and by O{sub 2} pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.

  10. Expression of Rhizobial Nitrogenase: Influence of Plant Cell-Conditioned Medium †

    PubMed Central

    Bednarski, Mary Ann; Reporter, Minocher

    1978-01-01

    Conditioned medium was obtained from suspension cultures of soybean (Glycine max L. Merrit) cells after incubating them for 4 to 8 days with rhizobia which were separated from the soybean cells by two dialysis bags, one within another. This conditioned medium from the plant cell side (PCM) of the two membranes was used to elicit and influence nitrogenase activity (acetylene reduction) in rhizobia. When conditions for obtaining PCM from the soybean cell suspension cultures were varied, it could be shown that freshly grown rhizobia were able to induce active compounds in the PCM. These compounds caused acetylene reduction activity in test rhizobia under conditions where control rhizobia, containing various substrates, showed little or no acetylene reduction activity. Rhizobia that were already capable of acetylene reduction could not induce such compounds in the PCM when this was included with test rhizobia. The PCM from soybean cultures was also found to aid the expression of nitrogenase activity in suspension cultures of rhizobia normally associated with either peas, lupins, broad beans, or clovers. This is the first communication indicating nitrogenase activity in freeliving cultures for various species of rhizobia. PMID:16345300

  11. Regulation of nitrogenase-2 in Azotobacter vinelandii by ammonium, molybdenum, and vanadium.

    PubMed

    Jacobitz, S; Bishop, P E

    1992-06-01

    Under diazotrophic conditions in the absence of molybdenum and in the presence of vanadium, Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase-2, a V-containing enzyme complex encoded by vnfH (the gene for dinitrogenase reductase-2), and vnfDGK (the genes for dinitrogenase-2 subunits). Accumulation of the vnfHorfFd and vnfDGK transcripts occurred under Mo-deficient conditions in the presence and absence of V; however, in the case of vnfDGK, the protein products only accumulated in the presence of V. This suggests that V is required for translation of the vnfDGK transcripts. In addition, expression of vnfH-lacZ and vnfD-lacZ transcriptional fusions was only partially repressed in the presence of NH4+. Transcripts hybridizing with vnfH (1.4 and 1.0 kb), vnfDG (3.4 and 1.8 kb), and vnfK (3.4 kb) were detected in RNA extracted from wild-type cells cultured with NH4+ in the presence or absence of V. However, nitrogenase-2 subunits were not detected in extracts of cells derepressed for nitrogenase-2 in the presence of NH4+. These results indicate that this nitrogen source acts at the posttranscriptional level as well as at the transcriptional level. vnf transcripts were not detected in the presence of Mo (with or without NH4+).

  12. About the role and underlying mechanisms of cofactors in anaphylaxis.

    PubMed

    Wölbing, F; Fischer, J; Köberle, M; Kaesler, S; Biedermann, T

    2013-09-01

    Anaphylaxis is the systemic and most severe presentation of type I allergy. A number of conditions were identified that modulate the onset of anaphylaxis such as co- or augmentation factors, which significantly lower the allergen dose necessary for triggering anaphylaxis. Next to physical exercise or alcohol consumption, co-administration of nonsteroidal anti-inflammatory drugs (NSAID) or concomitant infectious diseases are well-documented cofactors of anaphylaxis. Registries for anaphylaxis document a role for cofactors in about 30% of anaphylactic reactions. Some disease entities such as 'wheat-dependent exercise-induced anaphylaxis' (WDEIA) are explicitly characterized by elicitation of anaphylaxis only in the presence of at least one such cofactor. Using WDEIA as a model disease, studies demonstrated that exercise increases skin prick test reactivity to and bioavailability of the allergen. Additional data indicate that alcohol consumption and NSAID administration display similar effects. Modulation of the cellular activation threshold is another mechanism underlying cofactor-induced anaphylaxis, most likely also functional when infectious diseases orchestrate elicitation of anaphylaxis. Cofactors are increasingly accepted to play a fundamental role in eliciting anaphylaxis. Consequently, to improve patient management modalities, a better understanding of the underlying mechanisms is warranted. This review aims to update clinicians and clinical scientists on recent developments.

  13. Co-factors for smoking and evolutionary psychobiology.

    PubMed

    Pomerleau, C S

    1997-04-01

    Smoking is becoming increasingly concentrated in people with co-factors such as depression, attention deficit-hyperactivity disorder, anxiety disorders, and bulimia/bingeing. These behavioral or cognitive patterns may be adaptive or neutral in the conditions under which we evolved but maladaptive in environments requiring alertness for extended periods, where a fully mobilized fight-or-flight response is inappropriate, and where food availability makes lack of an "appestat" a liability. Such conditions are amenable to management by nicotine because of its ability to produce small but reliable adjustments in relevant cognitive and behavioral functions. Moreover, symptomatology may be unmasked or exacerbated by nicotine abstinence, persisting beyond the usual time-course for nicotine withdrawal, which may explain the particular attraction of smoking and the difficulty these individuals experience in quitting without necessarily requiring that they be more nicotine-dependent. The implications are: (1) a better understanding of the evolutionary psychobiology of smoking may promote development of tailored interventions for smokers with co-factors; (2) nicotine may have therapeutic applications for non-smokers with co-factors; (3) because smoking has a fairly high heritability index, and because of evidence of assortative mating, special prevention efforts targeting children of smokers with co-factors, as well as early identification of the co-factor itself, may be needed.

  14. Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

    PubMed Central

    Fallik, E; Chan, Y K; Robson, R L

    1991-01-01

    Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases

  15. 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.

  16. 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.

  17. 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

  18. Emerging roles for tubulin folding cofactors at the centrosome.

    PubMed

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

    2010-07-01

    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/G(2), a discovery that directly implicates tubulin cofactors in the cell division, cell migration and cell signaling research fields.

  19. A complex iron-calcium cofactor catalyzing phosphotransfer chemistry.

    PubMed

    Yong, Shee Chien; Roversi, Pietro; Lillington, James; Rodriguez, Fernanda; Krehenbrink, Martin; Zeldin, Oliver B; Garman, Elspeth F; Lea, Susan M; Berks, Ben C

    2014-09-01

    Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes, we have determined the structure of the widely occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active-site cofactor comprising two antiferromagnetically coupled ferric iron ions (Fe(3+)), three calcium ions (Ca(2+)), and an oxo group bridging three of the metal ions. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active-site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.

  20. Binding cofactors with triplex-based DNA motifs.

    PubMed

    Kröner, Christoph; Göckel, Anja; Liu, Wenjing; Richert, Clemens

    2013-11-18

    Cofactors are pivotal compounds for the cell and many biotechnological processes. It is therefore interesting to ask how well cofactors can be bound by oligonucleotides designed not to convert but to store and release these biomolecules. Here we show that triplex-based DNA binding motifs can be used to bind nucleotides and cofactors, including NADH, FAD, SAM, acetyl CoA, and tetrahydrofolate (THF). Dissociation constants between 0.1 μM for SAM and 35 μM for THF were measured. A two-nucleotide gap still binds NADH. The selectivity for one ligand over the others can be changed by changing the sequence of the binding pocket. For example, a mismatch placed in one of the two triplets adjacent to the base-pairing site changes the selectivity, favoring the binding of FAD over that of ATP. Further, changing one of the two thymines of an A-binding motif to cytosine gives significant affinity for G, whereas changing the other does not. Immobilization of DNA motifs gives beads that store NADH. Exploratory experiments show that the beads release the cofactor upon warming to body temperature.

  1. 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.

  2. 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

  3. Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis.

    PubMed

    Ajjawi, Imad; Rodriguez Milla, Miguel A; Cushman, John; Shintani, David K

    2007-09-01

    Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.

  4. 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

  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. Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor.

    PubMed

    Krzek, Marzena; van Beek, Hugo L; Permentier, Hjalmar P; Bischoff, Rainer; Fraaije, Marco W

    2016-01-01

    A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.

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

    PubMed

    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

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

    PubMed

    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

    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.

  9. New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (nifH) Genes

    PubMed Central

    Zehr, Jonathan P.; Mellon, Mark T.; Zani, Sabino

    1998-01-01

    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 α and γ 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. PMID:9726895

  10. 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

  11. 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.

  12. Study of the cofactor conditions: Conditions of supercompatibility between phases

    NASA Astrophysics Data System (ADS)

    Chen, Xian; Srivastava, Vijay; Dabade, Vivekanand; James, Richard D.

    2013-12-01

    The cofactor conditions, introduced in James and Zhang(2005), are conditions of compatibility between phases in martensitic materials. They consist of three subconditions: (i) the condition that the middle principal stretch of the transformation stretch tensor U is unity (λ2=1), (ii) the condition a·Ucof(U2-I)n=0, where the vectors a and n are certain vectors arising in the specification of the twin system, and (iii) the inequality trU2+det U2-(1/4)|a|2|n|2≥2. Together, these conditions are necessary and sufficient for the equations of the crystallographic theory of martensite to be satisfied for the given twin system but for any volume fraction f of the twins, 0≤f≤1. This contrasts sharply with the generic solutions of the crystallographic theory which have at most two such volume fractions for a given twin system of the form f* and 1-f*. In this paper we simplify the form of the cofactor conditions, we give their specific forms for various symmetries and twin types, we clarify the extent to which the satisfaction of the cofactor conditions for one twin system implies its satisfaction for other twin systems. In particular, we prove that the satisfaction of the cofactor conditions for either Type I or Type II twins implies that there are solutions of the crystallographic theory using these twins that have no elastic transition layer. We show that the latter further implies macroscopically curved, transition-layer-free austenite/martensite interfaces for Type I twins, and planar transition-layer-free interfaces for Type II twins which nevertheless permit significant flexibility (many deformations) of the martensite. We identify some real material systems nearly satisfying the cofactor conditions. Overall, the cofactor conditions are shown to dramatically increase the number of deformations possible in austenite/martensite mixtures without the presence of elastic energy needed for coexistence. In the context of earlier work that links the special case λ2

  13. Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

    PubMed Central

    Zehr, Jonathan P.; Limberger, Ronald J.; Ohki, Kaori; Fujita, Yoshihiko

    1990-01-01

    A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA. Images PMID:16348356

  14. Calorimetry of Nitrogenase-Mediated Reductions in Detached Soybean Nodules 1

    PubMed Central

    Heytler, Peter G.; Hardy, Ralph W. F.

    1984-01-01

    Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N2 fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made. There was major heat output by nodules for all of the nitrogenase substrates tested (H+, N2, N2O, and C2H2) further establishing the in vivo energy inefficiency of biological N2 fixation. Exposure to a short burst of 100% O2 partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H+ reductions was 171 ± 6 kilocalories per mole H2 evolved in an Ar-O2 atmosphere, that for N2 fixation was 784 ± 26 kilocalories per mole H2 evolved and N2 fixed, and that for C2H2 reduction was 250 ± 12 kilocalories/mole C2H4 formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e− is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N2 fixation cost of 9.5 grams glucose per gram N2 fixed or 3.8 grams C per gram N2, which is in close agreement with earlier calculations based on nodular CO2 production. PMID:16663616

  15. 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.

  16. 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.

  17. Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis.

    PubMed Central

    Kessler, P S; McLarnan, J; Leigh, J A

    1997-01-01

    We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 microM, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases. PMID:8990309

  18. Nitrite and Hydroxylamine as Nitrogenase Substrates: Mechanistic Implications for the Pathway of N2 Reduction

    PubMed Central

    2015-01-01

    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. PMID:25136926

  19. Nitrogenase switch-off by ammonium ions in Azospirillum brasilense requires the GlnB nitrogen signal-transducing protein.

    PubMed

    Klassen, Giseli; Souza, Emanuel M; Yates, M Geoffrey; Rigo, Liu Un; Costa, Roberta M; Inaba, Juliana; Pedrosa, Fábio O

    2005-09-01

    Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.

  20. 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.

  1. Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide.

    PubMed Central

    Lowe, D J; Fisher, K; Thorneley, R N

    1990-01-01

    The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme. PMID:2268290

  2. Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae.

    PubMed Central

    Madigan, M; Cox, S S; Stegeman, R A

    1984-01-01

    Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species. PMID:6581158

  3. Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

    PubMed

    Hom, S S; Graham, L A; Maier, R J

    1985-03-01

    Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.

  4. 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

  5. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii.

    PubMed Central

    Joerger, R D; Loveless, T M; Pau, R N; Mitchenall, L A; Simon, B H; Bishop, P E

    1990-01-01

    The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnfD (encoding the alpha subunit of dinitrogenase 2), vnfG (encoding a product similar to the delta subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the beta subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnfD contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vnfH-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnfH are still able to synthesize the alpha and beta subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium. A strain (RP1.11) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2. Images PMID:2345152

  6. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii.

    PubMed

    Joerger, R D; Loveless, T M; Pau, R N; Mitchenall, L A; Simon, B H; Bishop, P E

    1990-06-01

    The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnfD (encoding the alpha subunit of dinitrogenase 2), vnfG (encoding a product similar to the delta subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the beta subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnfD contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vnfH-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnfH are still able to synthesize the alpha and beta subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium. A strain (RP1.11) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.

  7. 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.

  8. Metabolic Pathways for Photobiological Hydrogen Production by Nitrogenase- and Hydrogenase-containing Unicellular Cyanobacteria Cyanothece*

    PubMed Central

    Skizim, Nicholas J.; Ananyev, Gennady M.; Krishnan, Anagha; Dismukes, G. Charles

    2012-01-01

    Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H2 effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H2 production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H2 is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O2 by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO2. Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H2 production rate by 2-fold (at the expense of the dark-H2 rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement. PMID:22128188

  9. The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

    PubMed Central

    Lowe, D J; Thorneley, R N

    1984-01-01

    A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein. PMID:6395861

  10. The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

    PubMed

    Lowe, D J; Thorneley, R N

    1984-12-15

    A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

  11. 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.

  12. 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. PMID:25850532

  13. Computation of cofactors of /sI-A/ with applications

    NASA Technical Reports Server (NTRS)

    Mitchell, J. R.; Nail, J. B.; Mcdaniel, W. L., Jr.

    1979-01-01

    An alternate approach for computing the determinant and the cofactors of the (sI-A) matrices is presented along with a numerical code for implementing this method with the aid of transforming a matrix to phase variable canonical form. Danilevskii's method was routinely used in this capacity for systems up to 26th order and it appears that this technique is also applicable to much higher order systems.

  14. 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.

  15. Cofactoring and Dimerization of Proteinase-Activated Receptors

    PubMed Central

    Lin, Huilan; Liu, Allen P.; Smith, Thomas H.

    2013-01-01

    Proteinase-activated receptors (PARs) are G protein–coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other’s activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling. PMID:24064459

  16. 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.

  17. MYC cofactors: molecular switches controlling diverse biological outcomes.

    PubMed

    Hann, Stephen R

    2014-06-17

    The transcription factor MYC has fundamental roles in proliferation, apoptosis, tumorigenesis, and stem cell pluripotency. Over the last 30 years extensive information has been gathered on the numerous cofactors that interact with MYC and the target genes that are regulated by MYC as a means of understanding the molecular mechanisms controlling its diverse roles. Despite significant advances and perhaps because the amount of information learned about MYC is overwhelming, there has been little consensus on the molecular functions of MYC that mediate its critical biological roles. In this perspective, the major MYC cofactors that regulate the various transcriptional activities of MYC, including canonical and noncanonical transactivation and transcriptional repression, will be reviewed and a model of how these transcriptional mechanisms control MYC-mediated proliferation, apoptosis, and tumorigenesis will be presented. The basis of the model is that a variety of cofactors form dynamic MYC transcriptional complexes that can switch the molecular and biological functions of MYC to yield a diverse range of outcomes in a cell-type- and context-dependent fashion.

  18. Magnetostructural dynamics of Rieske versus ferredoxin iron-sulfur cofactors.

    PubMed

    Ali, Md Ehesan; Staemmler, Volker; Marx, Dominik

    2015-03-01

    The local chemical environment of the [2Fe-2S] cofactor hosted by ferredoxin and Rieske-type proteins is fundamentally different due to the presence of distinct ligands at the two iron centers in the case of Rieske proteins, whereas they are identical in ferredoxins. This renders Rieske [2Fe-2S] cores chemically asymmetric and results in more complex vibrational spectra as compared to ferredoxin. Likewise, one would expect other properties, for instance the dynamics of the magnetic exchange coupling constant J, to be also more complex. Applying ab initio molecular dynamics using our recently introduced spin-constrained two-determinant extended broken symmetry (CEBS) approach to Rieske and ferredoxin model complexes at 300 K, we extract the molecular fluctuations and the resulting magnetostructural cross-correlations involving the antiferromagnetic exchange interaction J(t). This analysis demonstrates that the details of the magnetostructural dynamics are indeed distinctly different for Rieske and ferredoxin cofactors, while the time averages of 〈J〉 are shown to be essentially identical. In particular, the frequency window between about 200 and 350 cm(-1), is a "fingerprint region" that allows one to distinguish chemically asymmetric from symmetric cofactors and thus Rieske proteins from ferredoxins.

  19. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

    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.

  1. 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.

  2. 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

  3. Hydrogen Inhibition of Nitrogen Reduction by Nitrogenase in Isolated Soybean Nodule Bacteroids 1

    PubMed Central

    Rasche, Madeline E.; Arp, Daniel J.

    1989-01-01

    Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N2 reduction by nitrogenase. To evaluate the significance of H2 inhibition in vivo, we have measured the apparent inhibition constant for H2 inhibition of N2 reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N2 reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O2 and various concentrations of N2 and H2. The apparent inhibition constant for H2 under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H2 inhibition of N2 reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules. PMID:16667084

  4. Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings.

    PubMed

    Yanthan, Mhathung; Misra, Arvind K

    2013-11-01

    Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently. PMID:24287658

  5. 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

  6. Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii.

    PubMed

    Premakumar, R; Jacobson, M R; Loveless, T M; Bishop, P E

    1992-09-01

    Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms. PMID:1281443

  7. Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii.

    PubMed

    Premakumar, R; Jacobson, M R; Loveless, T M; Bishop, P E

    1992-09-01

    Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms.

  8. 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. PMID:12043002

  9. 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.

  10. Lanthanide ions as required cofactors for DNA catalysts.

    PubMed

    Dokukin, Victor; Silverman, Scott K

    2012-01-01

    We report that micromolar concentrations of lanthanide ions can be required cofactors for DNA-hydrolyzing deoxyribozymes. Previous work identified deoxyribozymes that simultaneously require both Zn(2+) and Mn(2+) to achieve DNA-catalyzed DNA hydrolysis (10(12) rate enhancement); a mutant of one such DNA catalyst requires only Zn(2+). Here we show that in vitro selection in the presence of 10 µM lanthanide ion (Ce(3+), Eu(3+), or Yb(3+)) along with 1 mM Zn(2+) leads to numerous DNA-hydrolyzing deoxyribozymes that strictly require the lanthanide ion as well as Zn(2+) for catalytic activity. These DNA catalysts have a range of lanthanide dependences, including some deoxyribozymes that strongly favor one particular lanthanide ion (e.g., Ce(3+) > Eu(3+) > Yb(3+)) and others that function well with more than one lanthanide ion. Intriguingly, two of the Yb(3+)-dependent deoxyribozymes function well with Yb(3+) alone (K(d,app) ~10 µM, in the absence of Zn(2+)) and have little or no activity with Eu(3+) or Ce(3+). In contrast to these selection outcomes when lanthanide ions were present, new selections with Zn(2+) or Mn(2+) alone, or Zn(2+) with Mg(2+)/Ca(2+), led primarily to deoxyribozymes that cleave DNA by deglycosylation and β-elimination rather than by hydrolysis, including several instances of depyrimidination. We conclude that lanthanide ions warrant closer attention as cofactors when identifying new nucleic acid catalysts, especially for applications in which high concentrations of polyvalent metal ion cofactors are undesirable.

  11. 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. PMID:25954742

  12. 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

  13. Effect of Aptamer Binding on the Electron-Transfer Properties of Redox Cofactors.

    PubMed

    Emahi, Ismaila; Gruenke, Paige R; Baum, Dana A

    2015-12-01

    In vitro selection or SELEX has allowed for the identification of functional nucleic acids (FNAs) that can potentially mimic and replace protein enzymes. These FNAs likely interact with cofactors, just like enzymes bind cofactors in their active sites. Investigating how FNA binding affects cofactor properties is important for understanding how an active site is formed and for developing useful enzyme mimics. Oxidoreductase enzymes contain cofactors in their active sites that allow the enzymes to do redox chemistry. In certain applications, these redox cofactors act as electron-transfer shuttles that transport electrons between the enzymes' active sites and electrode surfaces. Three redox cofactors commonly found in oxidoreductases are flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD(+)), and pyrroloquinoline quinone (PQQ). We are interested in investigating how DNA aptamers that bind these cofactors influence the cofactors' redox abilities and if these aptamer-cofactor complexes could serve as redox catalysts. We employed cyclic voltammetry and amperometry to study the electrochemical properties of NAD(+) and PQQ when bound to DNA aptamers. Our results suggest that the aptamers provide a stable environment for the cofactor to participate in redox reactions, although enhanced redox activity was not observed. This work provides a foundation for the development of new FNAs capable of redox activity.

  14. Coordinated expression of fdxD and molybdenum nitrogenase genes promotes nitrogen fixation by Rhodobacter capsulatus in the presence of oxygen.

    PubMed

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

    2014-02-01

    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.

  15. 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.

  16. Lanthanide cofactors accelerate DNA-catalyzed synthesis of branched RNA.

    PubMed

    Javadi-Zarnaghi, Fatemeh; Höbartner, Claudia

    2013-08-28

    Most deoxyribozymes (DNA catalysts) require metal ions as cofactors for catalytic activity, with Mg(2+), Mn(2+), and Zn(2+) being the most represented activators. Trivalent transition-metal ions have been less frequently considered. Rare earth ions offer attractive properties for studying metal ion binding by biochemical and spectroscopic methods. Here we report the effect of lanthanide cofactors, in particular terbium (Tb(3+)), for DNA-catalyzed synthesis of 2',5'-branched RNA. We found up to 10(4)-fold increased ligation rates for the 9F7 deoxribozyme using 100 μM Tb(3+) and 7 mM Mg(2+), compared to performing the reaction with 7 mM Mg(2+) alone. Combinatorial mutation interference analysis (CoMA) was used to identify nucleotides in the catalytic region of 9F7 that are essential for ligation activity with different metal ion combinations. A minimized version of the DNA enzyme sustained high levels of Tb(3+)-assisted activity. Sensitized luminescence of Tb(3+) bound to DNA in combination with DMS probing and DNase I footprinting results supported the CoMA data. The accelerating effect of Tb(3+) was confirmed for related RNA-ligating deoxyribozymes, pointing toward favorable activation of internal 2'-OH nucleophiles. The results of this study offer fundamental insights into nucleotide requirements for DNA-catalyzed RNA ligation and will be beneficial for practical applications that utilize 2',5'-branched RNA.

  17. HMGB1 is a cofactor in mammalian base excision repair.

    PubMed

    Prasad, Rajendra; Liu, Yuan; Deterding, Leesa J; Poltoratsky, Vladimir P; Kedar, Padmini S; Horton, Julie K; Kanno, Shin-Ichiro; Asagoshi, Kenjiro; Hou, Esther W; Khodyreva, Svetlana N; Lavrik, Olga I; Tomer, Kenneth B; Yasui, Akira; Wilson, Samuel H

    2007-09-01

    Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells. PMID:17803946

  18. Mechanism by which metal cofactors control substrate specificity in pyrophosphatase.

    PubMed Central

    Zyryanov, Anton B; Shestakov, Alexander S; Lahti, Reijo; Baykov, Alexander A

    2002-01-01

    Family I soluble pyrophosphatases (PPases) exhibit appreciable ATPase activity in the presence of a number of transition metal ions, but not the physiological cofactor Mg(2+). The results of the present study reveal a strong correlation between the catalytic efficiency of three family I PPases (from Saccharomyces cerevisiae, Escherichia coli and rat liver) and one family II PPase (from Streptococcus mutans ) in ATP and tripolyphosphate (P(3)) hydrolysis in the presence of Mg(2+), Mn(2+), Zn(2+) and Co(2+) on the one hand, and the phosphate-binding affinity of the enzyme subsite P2 that interacts with the electrophilic terminal phosphate group of ATP on the other. A similar correlation was observed in S. cerevisiae PPase variants with modified P1 and P2 subsites. The effect of the above metal ion cofactors on ATP binding to S. cerevisiae PPase paralleled their effect on phosphate binding, resulting in a low affinity of Mg-PPase to ATP. We conclude that PPase mainly binds ATP and P(3) through the terminal phosphate group that is attacked by water. Moreover, this interaction is critical in creating a reactive geometry at the P2 site with these bulky substrates, which do not otherwise fit the active site perfectly. We propose further that ATP is not hydrolysed by Mg-PPase, since its interaction with the terminal phosphate is not adequately strong for proper positioning of the nucleophile-electrophile pair. PMID:12169093

  19. 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.

  20. Hox Specificity: Unique Roles for Cofactors and Collaborators

    PubMed Central

    Mann, Richard S.; Lelli, Katherine M.; Joshi, Rohit

    2010-01-01

    Hox proteins are well known for executing highly specific functions in vivo, but our understanding of the molecular mechanisms underlying gene regulation by these fascinating proteins has lagged behind. The premise of this review is that an understanding of gene regulation — by any transcription factor—requires the dissection of the cis-regulatory elements that they act upon. With this goal in mind, we review the concepts and ideas regarding gene regulation by Hox proteins and apply them to a curated list of directly regulated Hox cis-regulatory elements that have been validated in the literature. Our analysis of the Hox-binding sites within these elements suggests several emerging generalizations. We distinguish between Hox cofactors, proteins that bind DNA cooperatively with Hox proteins and thereby help with DNA-binding site selection, and Hox collaborators, proteins that bind in parallel to Hox-targeted cis-regulatory elements and dictate the sign and strength of gene regulation. Finally, we summarize insights that come from examining five X-ray crystal structures of Hox-cofactor-DNA complexes. Together, these analyses reveal an enormous amount of flexibility into how Hox proteins function to regulate gene expression, perhaps providing an explanation for why these factors have been central players in the evolution of morphological diversity in the animal kingdom. PMID:19651302

  1. Regulation of the DNA Damage Response by p53 Cofactors

    PubMed Central

    Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2012-01-01

    The selective expression of p53-targeted genes is central to the p53-mediated DNA damage response. It is affected by multiple factors including posttranslational modifications and cofactors of p53. Here, we proposed an integrated model of the p53 network to characterize how the cellular response is regulated by key cofactors of p53, Hzf and ASPP. We found that the sequential induction of Hzf and ASPP is crucial to a reliable cell-fate decision between survival and death. After DNA damage, activated p53 first induces Hzf, which promotes the expression of p21 to arrest the cell cycle and facilitate DNA repair. The cell recovers to normal proliferation after the damage is repaired. If the damage is beyond repair, Hzf is effectively degraded, and activated E2F1 induces ASPP, which promotes the expression of Bax to trigger apoptosis. Furthermore, interrupting the induction of Hzf or ASPP remarkably impairs the cellular function. We also proposed two schemes for the production of the unknown E3 ubiquitin ligase for Hzf degradation: it is induced by either E2F1 or p53. In both schemes, the sufficient degradation of Hzf is required for apoptosis induction. These results are in good agreement with experimental observations or are experimentally testable. PMID:22677378

  2. 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.

  3. Heparin cofactor II is degraded by heparan sulfate and dextran sulfate.

    PubMed

    Saito, Akio

    2015-02-20

    Heparan sulfate normally binds to heparin cofactor II and modulates the coagulation pathway by inhibiting thrombin. However, when human heparin cofactor II was incubated with heparan sulfate, heparin cofactor II became degraded. Other glycosaminoglycans were tested, including hyaluronic acid, chondroitin sulfates, dermatan sulfate, and heparin, but only dextran sulfate also degraded heparin cofactor II. Pretreatment of heparan sulfate with heparinase reduced its heparin cofactor II-degrading activity. Heparan sulfate and dextran sulfate diminished the thrombin inhibitory activity of heparin cofactor II. Other serpins, including antithrombin III and pigment epithelium-derived factor, were also degraded by heparan sulfate. This is the first evidence of acidic polysaccharides exhibiting protein-degrading activity without the aid of other proteins.

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

    PubMed

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

    2016-07-15

    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 Fdc1(Ubix), 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.

  5. 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.

  6. Cofactory: sequence-based prediction of cofactor specificity of Rossmann folds.

    PubMed

    Geertz-Hansen, Henrik Marcus; Blom, Nikolaj; Feist, Adam M; Brunak, Søren; Petersen, Thomas Nordahl

    2014-09-01

    Obtaining optimal cofactor balance to drive production is a challenge in metabolically engineered microbial production strains. To facilitate identification of heterologous enzymes with desirable altered cofactor requirements from native content, we have developed Cofactory, a method for prediction of enzyme cofactor specificity using only primary amino acid sequence information. The algorithm identifies potential cofactor binding Rossmann folds and predicts the specificity for the cofactors FAD(H2), NAD(H), and NADP(H). The Rossmann fold sequence search is carried out using hidden Markov models whereas artificial neural networks are used for specificity prediction. Training was carried out using experimental data from protein-cofactor structure complexes. The overall performance was benchmarked against an independent evaluation set obtaining Matthews correlation coefficients of 0.94, 0.79, and 0.65 for FAD(H2), NAD(H), and NADP(H), respectively. The Cofactory method is made publicly available at http://www.cbs.dtu.dk/services/Cofactory.

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

    PubMed

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

    2015-11-05

    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.

  8. 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

  9. 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.; Sadler, Natalie C.; Melnicki, Matthew R.; Hill, Eric A.; Markillie, Lye Meng; Nicora, Carrie D.; Wright, Aaron T.; Romine, Margaret F.; et al

    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

  10. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

    PubMed Central

    Joerger, R D; Jacobson, M R; Premakumar, R; Wolfinger, E D; Bishop, P E

    1989-01-01

    The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3. Images PMID:2644222

  11. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

    PubMed

    Joerger, R D; Jacobson, M R; Premakumar, R; Wolfinger, E D; Bishop, P E

    1989-02-01

    The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3. PMID:2644222

  12. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

    PubMed

    Joerger, R D; Jacobson, M R; Premakumar, R; Wolfinger, E D; Bishop, P E

    1989-02-01

    The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

  13. Effect of the over-expression of PII and PZ proteins on the nitrogenase activity of Azospirillum brasilense.

    PubMed

    Huergo, Luciano F; Filipaki, Angela; Chubatsu, Leda S; Yates, M Geoffrey; Steffens, Maria Berenice; Pedrosa, Fabio O; Souza, Emanuel M

    2005-12-01

    The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

  14. Effects of long-term preservation of Frankia strains on infectivity, effectivity, and in vitro nitrogenase activity

    SciTech Connect

    Fontaine, M.S.; Young, P.H.; Torrey, J.G.

    1986-04-01

    Frankia strain HFP ArI3 which had been preserved for 27 months by being lyophilized, frozen in glycerol, or stored in complex medium was successfully used as an inoculum after being subcultured for inducing nodulation and nitrogen fixation of Alnus rubra. Glycerol-preserved HFPArI3 produced significantly lower rates of nitrogenase activity than did lyophilized or complex-medium-preserved inocula. Bacteria that had been preserved by all three methods were successfully induced to fix atmospheric nitrogen by being cultured in nitrogen-free medium. Subculturing of these cells in nitrogen-free medium a second and third time yielded increasing rates of nitrogenase activity. Initial nitrogenase activity was detected on days 5, 4, and 3 during the first, second, and third subcultures after preservation, respectively. Maximum activity was observed on days 11, 10, and 8 during the first, second, and third subcultures, respectively. A description is given of standard culture techniques used in our laboratory for Frankia isolates, and methods used to distribute Frankia cultures by mail are described.

  15. 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

  16. 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.

  17. NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

    SciTech Connect

    Dodsworth, Jeremy A.; Leigh, John A.

    2007-12-14

    Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI{sub 1,2} binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI{sub 1,2} binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI{sub 1,2}, decreasing its inhibitory effect. NifI{sub 1,2} prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI{sub 1,2} was unable to bind to an AlF{sub 4}{sup -}-stabilized Fe protein:MoFe protein complex. NifI{sub 1,2} inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI{sub 1,2} competes with the Fe protein for binding to MoFe protein and prevents electron transfer.

  18. Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

    PubMed Central

    Mylona, P V; Premakumar, R; Pau, R N; Bishop, P E

    1996-01-01

    In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions. PMID:8550418

  19. Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

    PubMed

    Mylona, P V; Premakumar, R; Pau, R N; Bishop, P E

    1996-01-01

    In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

  20. Experimental Determination of the Respiration Associated with Soybean/Rhizobium Nitrogenase Function, Nodule Maintenance, and Total Nodule Nitrogen Fixation 1

    PubMed Central

    Rainbird, Ross M.; Hitz, William D.; Hardy, Ralph W. F.

    1984-01-01

    The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function. Rates of CO2 evolution and H2 evolution from intact, nodulated root systems under Ar:O2 atmospheres decreased in parallel when plants were maintained in an extended dark period. While H2 evolution approached zero after 36 hours of darkness at 22°C, CO2 evolution rate remained at 38° of the rate measured in light. Of the remaining CO2 evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO2 (per gram dry weight nodule) per hour at 22°C and 30°C, respectively. The cost of N2 fixation in terms of CO2 evolved per electron pair utilized by nitrogenase was estimated from the slope of H2 evolution rate versus CO2 evolution rate. The cost was 2 moles CO2 evolved per mole H2 evolved and was independent of temperature. In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H2 evolution. PMID:16663599

  1. Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein.

    PubMed

    Miller, R W; Eady, R R

    1989-11-01

    The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.

  2. 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

  3. Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors.

    PubMed

    Jauker, Mario; Griesser, Helmut; Richert, Clemens

    2015-11-23

    How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD(+), FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation.

  4. Parallel texture structures with cofactor zeros in lepton sector

    NASA Astrophysics Data System (ADS)

    Wang, Weijian

    2014-06-01

    In this paper we investigate the parallel texture structures with cofactor zeros in the charged lepton and neutrino sectors. The textures cannot be obtained from arbitrary leptonic matrices by making weak basis transformations, which therefore have physical meaning. The 15 parallel textures are grouped as 4 classes where each class has the same physical implications. It is founded that one of them is not phenomenologically viable and another is equivalent to the texture zero structures extensively explored in previous literature. Thus we focus on the other two classes of parallel texture structures and study the their phenomenological implications. The constraints on the physical variables are obtained for each class, which is essential for the model selection and can be measured by future experiments. The model realization is illustrated in a radiated lepton mass model.

  5. 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.

  6. Parallel lepton mass matrices with texture and cofactor zeros

    NASA Astrophysics Data System (ADS)

    Wang, Weijian

    2014-08-01

    In this paper we investigate the parallel texture structures containing texture zeros in the charged lepton mass matrix Ml and cofactor zeros in the neutrino mass matrix Mν. These textures are interesting since they are related to the Zn flavor symmetries. Using the weak basis permutation transformation, the 15 parallel textures are grouped as 4 classes (class I, II, III, and IV), with the matrices in each class sharing the same physical implications. Under the current experimental data, the classes I and III with inverted mass hierarchy and class II with normal mass hierarchy are phenomenologically acceptable. The correlations between some important physical variables are presented, which are essential for the model selection and can be tested by future experiments. The model realization is illustrated by means of Z4×Z2 flavor symmetry.

  7. Glucocorticoid receptor co-factors as therapeutic targets

    PubMed Central

    Simons, S. Stoney

    2010-01-01

    Summary Numerous transcriptional cofactors (e.g., coactivators, corepressors, and comodulators) are known to alter the maximal transcriptional activity (Amax) in gene induction and repression by steroid receptors in general and glucocorticoids in particular. However, recent data advance the earlier reports that these same factors also modify other parameters of glucocorticoid receptor transcriptional activity: the potency of agonists (or EC50) and the partial agonist activity of antisteroids (or PAA). In several instances, factors modulate the EC50 and/or PAA without changing Amax. Thus, studies of all three parameters reveal new factors acting at various stages of receptor action, thereby increasing the potential therapeutic targets for adjusting GR actions in pathological situations. PMID:20801081

  8. 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.

  9. Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors

    PubMed Central

    Jauker, Mario; Griesser, Helmut; Richert, Clemens

    2015-01-01

    How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD+, FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation. PMID:26435376

  10. 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

  11. 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.

  12. 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.

  13. 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. PMID:26152701

  14. 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. PMID:26192769

  15. Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.

    PubMed

    Saari, L L; Pope, M R; Murrell, S A; Ludden, P W

    1986-04-15

    Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity. PMID:3082874

  16. 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.

  17. Oxidative inactivation of the molybdenum-iron-protein component of nitrogenase from clostridium pasteurianum.

    PubMed

    Gomez-Moreno, C; Ke, B

    1979-07-31

    The sensitivity of the molybdenum-iron(MoFe)-protein of Clostridium pasteurianum nitrogenase toward oxidation has been studied by determining the enzymatic activity of this component after incubating it anaerobically in ferricyanide solutions of various oxidizing strengths (as measured by their oxidation potentials). It was found that the MoFe-protein remains active at potentials up to +350 mV (vs. standard hydrogen electrode) but becomes readily inactivated at more oxidizing potentials, after a lag period, depending on the potential level and temperature. Oxidative inactivation by ferricyanide results in the release of most of the Mo, Fe and S atoms from the protein which causes the loss of the absorption bands in the visible region. The metals and sulfur could be re-incorporated by incubation in a mixture containing thiol, sulfide, molybdate, and ferric iron. The EPR spectrum of the oxidatively inactivated MoFe-protein showed that both the high- and low-field signals are readily affected. Re-incorporation of the metals and sulfur into the "bleached" protein produced an EPR spectrum similar to that of the air-inactivated protein. Incubation of the Mo-Fe-protein with mersalyl abolished its enzymic activity. The difference spectrum before and after mersalyl treatment resembles that of the soluble spinach ferredoxin.

  18. 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

  19. 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.

  20. 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.

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

    PubMed

    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

  2. Kinetic Isotope Effects as a Probe of Hydrogen Transfers to and from Common Enzymatic Cofactors

    PubMed Central

    Roston, Daniel; Islam, Zahidul; Kohen, Amnon

    2013-01-01

    Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer. PMID:24161942

  3. Atomistic and Coarse Grain Topologies for the Cofactors Associated with the Photosystem II Core Complex.

    PubMed

    de Jong, Djurre H; Liguori, Nicoletta; van den Berg, Tom; Arnarez, Clement; Periole, Xavier; Marrink, Siewert J

    2015-06-25

    Electron transfers within and between protein complexes are core processes of the electron transport chains occurring in thylakoid (chloroplast), mitochondrial, and bacterial membranes. These electron transfers involve a number of cofactors. Here we describe the derivation of molecular mechanics parameters for the cofactors associated with the function of the photosystem II core complex: plastoquinone, plastoquinol, heme b, chlorophyll A, pheophytin, and β-carotene. Parameters were also obtained for ubiquinol and ubiquinone, related cofactors involved in the respiratory chain. Parameters were derived at both atomistic and coarse grain (CG) resolutions, compatible with the building blocks of the GROMOS united-atom and Martini CG force fields, respectively. Structural and thermodynamic properties of the cofactors were compared to experimental values when available. The topologies were further tested in molecular dynamics simulations of the cofactors in their physiological environment, e.g., either in a lipid membrane environment or in complex with the heme binding protein bacterioferritin.

  4. 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

    2013-07-01

    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.

  5. Kinetic isotope effects as a probe of hydrogen transfers to and from common enzymatic cofactors.

    PubMed

    Roston, Daniel; Islam, Zahidul; Kohen, Amnon

    2014-02-15

    Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer.

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

    DOE PAGES

    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 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

  7. 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.

  8. 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.

  9. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis.

    PubMed

    Hover, Bradley M; Tonthat, Nam K; Schumacher, Maria A; Yokoyama, Kenichi

    2015-05-19

    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, 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. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

  10. 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.

  11. Effect of pancreatic biliary reflux as a cofactor in cholecystitis.

    PubMed

    Amr, Abdel Raouf; Hamdy, Hussam Mohamed; Nasr, Magid Mahmoud; Hedaya, Mohammed Saied; Hassan, Ahmed Mohamed Abdelaziz

    2012-04-01

    This study assessed the effect of pancreatico-biliary reflux (PBR) as co-factor in the process of chronic cholecystitis by measurement of the levels of active pancreatic enzyme amylase in gallbladder bile and serum of patients undergoing cholecystectomy. Pancreatic Amylase levels in bile from the gallbladder and serum were measured during surgery in 68 patients with chronic calcular cholecystitis subjected to elective open or laparoscopic cholecystectomy in the National Hepatology and Tropical Medicine Research Institution and Theodore Bilharz Research Institute. Bile amylase was detected in 64 patients (94.1%) indicating pancreatico-biliary reflux. Biliary amylase level ranged from 20-50 IU/L in 42 patients (61.76%), below 20 IU/l in 14 patients (20.59%), over 50 IU/L in 8 patients (11.76%) and undetectable in two patients. According to gallbladder bile amylase, the incidence of Occult PBR in patients operated upon for chronic calcular cholecystitis was 94.1%. The reason should be clarified by further research and wider scale study. Routinely investigating biliary amylase in every patient having cholecystitis can be a method for early detection of precancerous lesions.

  12. Solvent-Dependent Pyranopterin Cyclization in Molybdenum Cofactor Model Complexes.

    PubMed

    Williams, Benjamin R; Gisewhite, Douglas; Kalinsky, Anna; Esmail, Alisha; Burgmayer, Sharon J Nieter

    2015-09-01

    The conserved pterin dithiolene ligand that coordinates molybdenum (Mo) in the cofactor (Moco) of mononuclear Mo enzymes can exist in both a tricyclic pyranopterin dithiolene form and as a bicyclic pterin-dithiolene form as observed in protein crystal structures of several bacterial molybdoenzymes. Interconversion between the tricyclic and bicyclic forms via pyran scission and cyclization has been hypothesized to play a role in the catalytic mechanism of Moco. Therefore, understanding the interconversion between the tricyclic and bicyclic forms, a type of ring-chain tautomerism, is an important aspect of study to understand its role in catalysis. In this study, equilibrium constants (K(eq)) as well as enthalpy, entropy, and free energy values are obtained for pyran ring tautomerism exhibited by two Moco model complexes, namely, (Et4N)[Tp*Mo(O)(S2BMOPP)] (1) and (Et4N)[Tp*Mo(O)(S2PEOPP)] (2), as a solvent-dependent equilibrium process. Keq values obtained from (1)H NMR data in seven deuterated solvents show a correlation between solvent polarity and tautomer form, where solvents with higher polarity parameters favor the pyran form.

  13. 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.

  14. Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases.

    PubMed

    Raverdy, Sylvine; Zhang, Yinhua; Foster, Jeremy; Carlow, Clotilde K S

    2007-12-01

    Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes.

  15. 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.

  16. Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

    PubMed Central

    Thiel, T

    1993-01-01

    Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis. Images PMID:8407800

  17. Regulation of nitrogenase gene expression by transcript stability in the cyanobacterium Anabaena variabilis.

    PubMed

    Pratte, Brenda S; Thiel, Teresa

    2014-10-01

    The nitrogenase gene cluster in cyanobacteria has been thought to comprise multiple operons; however, in Anabaena variabilis, the promoter for the first gene in the cluster, nifB1, appeared to be the primary promoter for the entire nif cluster. The structural genes nifHDK1 were the most abundant transcripts; however, their abundance was not controlled by an independent nifH1 promoter, but rather, by RNA processing, which produced a very stable nifH1 transcript and a moderately stable nifD1 transcript. There was also no separate promoter for nifEN1. In addition to the nifB1 promoter, there were weak promoters inside the nifU1 gene and inside the nifE1 gene, and both promoters were heterocyst specific. In an xisA mutant, which effectively separated promoters upstream of an 11-kb excision element in nifD1 from the downstream genes, the internal nifE1 promoter was functional. Transcription of the nif1 genes downstream of the 11-kb element, including the most distant genes, hesAB1 and fdxH1, was reduced in the xisA mutant, indicating that the nifB1 promoter contributed to their expression. However, with the exception of nifK1 and nifE1, which had no expression, the downstream genes showed low to moderate levels of transcription in the xisA mutant. The hesA1 gene also had a promoter, but the fdxH gene had a processing site just upstream of the gene. The processing of transcripts at sites upstream of nifH1 and fdxH1 correlated with increased stability of these transcripts, resulting in greater amounts than transcripts that were not close to processing sites. PMID:25092030

  18. Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum.

    PubMed

    Kern, M; Klipp, W; Klemme, J H

    1994-06-01

    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 H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) 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 H(2) 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. PMID:16349271

  19. 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

  20. Androgen receptor cofactors in prostate cancer: potential therapeutic targets of castration-resistant prostate cancer.

    PubMed

    Shiota, Masaki; Yokomizo, Akira; Fujimoto, Naohiro; Naito, Seiji

    2011-09-01

    Androgens, acting through the androgen receptor (AR), are responsible for many male reproductive and nonreproductive functions. Moreover, aberrant androgen/AR signaling plays a critical role in androgen-dependent prostate cancer (PCa) as well as castration-resistant prostate cancer (CRPC). The formation of a productive AR transcriptional complex requires AR cofactors that interact functionally and structurally with the AR. Since the discovery of the first such cofactor in 1995, an ever increasing number of proteins have been identified as AR coactivators or corepressors. The expression and function of several AR cofactors have been investigated in PCa, and a clear link between AR cofactors and the development and progression of PCa has been identified. Recently, AR splice variants in CRPC were reported, which display significant constitutive activity in the absence of ligand. Then, this discovery revolutionized the concept of AR cofactors in CRPC. The current review aims to provide an overview of AR cofactor proteins in the context of PCa. In addition, we discuss the potential of AR cofactors as novel therapeutic targets for PCa, particularly for CRPC.

  1. Dynamic expression of LIM cofactors in the developing mouse neural tube.

    PubMed

    Ostendorff, Heather P; Tursun, Baris; Cornils, Kerstin; Schlüter, Anne; Drung, Alexander; Güngör, Cenap; Bach, Ingolf

    2006-03-01

    The developmental regulation of LIM homeodomain transcription factors (LIM-HD) by the LIM domain-binding cofactors CLIM/Ldb/NLI and RLIM has been demonstrated. Whereas CLIM cofactors are thought to be required for at least some of the in vivo functions of LIM-HD proteins, the ubiquitin ligase RLIM functions as a negative regulator by its ability to target CLIM cofactors for proteasomal degradation. In this report, we have investigated and compared the protein expression of both factors in the developing mouse neural tube. We co-localize both proteins in many tissues and, although widely expressed, we detect high levels of both cofactors in specific neural tube regions, e.g., in the ventral neural tube, where motor neurons reside. The mostly ubiquitous distribution of RLIM- and CLIM-encoding mRNA differs from the more specific expression of both cofactors at the protein level, indicating post-transcriptional regulation. Furthermore, we show that both cofactors not only co-localize with each other but also with Isl and Lhx3 LIM-HD proteins in developing ventral neural tube neurons. Our results demonstrate the dynamic expression of cofactors participating in the regulation of LIM-HD proteins during the development of the neural tube in mice and suggest additional post-transcriptional regulation in the nuclear LIM-HD protein network.

  2. Structural insights into domain movement and cofactor specificity of glutamate dehydrogenase from Corynebacterium glutamicum.

    PubMed

    Son, Hyeoncheol Francis; Kim, Il-Kwon; Kim, Kyung-Jin

    2015-04-10

    Glutamate dehydrogenase (GDH) is an enzyme involved in the synthesis of amino acids by converting glutamate to α-ketoglutarate, and vice versa. To investigate the molecular mechanism of GDH, we determined a crystal structure of the Corynebacterium glutamicum-derived GDH (CgGDH) in complex with its NADP cofactor and α-ketoglutarate substrate. CgGDH functions as a hexamer, and each CgGDH monomer comprises 2 separate domains; a Rossmann fold cofactor-binding domain and a substrate-binding domain. The structural comparison between the apo- and cofactor/substrate-binding forms revealed that the CgGDH enzyme undergoes a domain movement during catalysis. In the apo-form, CgGDH exists as an open state, and upon binding of the substrate and cofactor the protein undergoes a conformation change to a closed state. Our structural study also revealed that CgGDH has cofactor specificity for NADP, but not NAD, and this was confirmed by GDH activity measurements. Residues involved in the stabilization of the NADP cofactor and the α-ketoglutarate substrate were identified, and their roles in substrate/cofactor binding were confirmed by site-directed mutagenesis experiments.

  3. Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions.

    PubMed

    Deleault, Nathan R; Walsh, Daniel J; Piro, Justin R; Wang, Fei; Wang, Xinhe; Ma, Jiyan; Rees, Judy R; Supattapone, Surachai

    2012-07-10

    Prions containing misfolded prion protein (PrP(Sc)) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrP(Sc) structure accompanied by a reduction in specific infectivity of >10(5)-fold, to undetectable levels, despite the ability of adapted "protein-only" PrP(Sc) molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrP(Sc) structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrP(Sc) conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.

  4. 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

  5. Role of a Ferredoxin Gene Cotranscribed with the nifHDK Operon in N2 Fixation and Nitrogenase “Switch-Off” of Azoarcus sp. Strain BH72

    PubMed Central

    Egener, Tanja; Martin, Dietmar E.; Sarkar, Abhijit; Reinhold-Hurek, Barbara

    2001-01-01

    The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a ς54-type promoter. Shorter transcripts sequentially missing genes of the 3′ part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% ± 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid “switch-off” of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be

  6. 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.

  7. Genetic variants and evolutionary analyses of heparin cofactor II.

    PubMed

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Goswami, Chandan

    2014-09-01

    Heparin cofactor II (HCII) belongs to serpin superfamily and it acts as a thrombin inhibitor in the coagulation cascade, in a glycosaminoglycan-dependent pathway using the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. This serpin belongs to multiple member group V2 of vertebrate serpin classification. However, there is no comprehensive study illustrating the exact phylogenetic history of HCII, to date. Herein, we explored phylogenetic traits of HCII genes. Structures of HCII gene from selected ray-finned fishes and lamprey varied in exon I and II with insertions of novel introns of which one in core domain for ray-finned fishes in exon II at the position 241c. We found HCII remain nested in the largest intron of phosphatidylinositol (PI) 4-kinase (PIK4CA) gene (genetic variants of this gene cause schizophrenia) at the origin of vertebrates, dated about 500MY old. We found that sequence features such as two acidic repeats (AR1-II), GAG-binding helix-D, three serpin motifs and inhibitory reactive center loop (RCL) of HCII protein are highly conserved in 55 vertebrates analyzed. We identified 985 HCII variants by analysis of 1092 human genomes with top three variation classes belongs to SNPs (84.3%), insertion (7.1%) and deletion (5.0%). We identified 37 deleterious mutations in the human HCII protein and we have described these mutations in relation to HCII sequence-structure-function relationships. These understandings may have clinical and medical importance as well.

  8. Partial purification and characterization of contact activation cofactor.

    PubMed Central

    Schiffman, S; Lee, P

    1975-01-01

    The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56 degrees C; gradually loses activity at 80 degrees C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by A1(OH)3; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 56 degrees C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XIa generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated. PMID:1184736

  9. 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

  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.

  11. Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases.

    PubMed

    Hayashi, Junji; Inoue, Shota; Kim, Kwang; Yoneda, Kazunari; Kawarabayasi, Yutaka; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2015-07-08

    NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.

  12. Cofactor mobility determines reaction outcome in the IMPDH/GMPR (β/α)8 barrel enzymes

    PubMed Central

    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

    2015-01-01

    IMP dehydrogenase (IMPDH) and GMP 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/GMPR family have not been identified. Here, we show that the GMPR reaction utilizes the same intermediate E-XMP* as IMPDH, but this 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 mimic 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 where the cofactor is 6 Å from IMP. Mutagenesis, substrate/cofactor analog experiments demonstrate that the “out” conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery activating ammonia. PMID:22037469

  13. Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

    PubMed

    Breitenbach, J; Zhu, C; Sandmann, G

    2001-11-01

    Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

  14. Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins.

    PubMed

    Ji, Hong-Fang; Chen, Lei; Zhang, Hong-Yu

    2008-08-01

    Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre-protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website.

  15. 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.

  16. 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.

  17. 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. PMID:25686600

  18. Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions.

    PubMed

    Wang, Tianwen David; Ma, Fei; Ma, Xingyuan; Wang, Ping

    2015-04-01

    Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.

  19. Altered cofactor regulation with disease-associated p97/VCP mutations.

    PubMed

    Zhang, Xiaoyi; Gui, Lin; Zhang, Xiaoyan; Bulfer, Stacie L; Sanghez, Valentina; Wong, Daniel E; Lee, YouJin; Lehmann, Lynn; Lee, James Siho; Shih, Pei-Yin; Lin, Henry J; Iacovino, Michelina; Weihl, Conrad C; Arkin, Michelle R; Wang, Yanzhuang; Chou, Tsui-Fen

    2015-04-01

    Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.

  20. Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

    PubMed

    Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

    2013-11-01

    Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

  1. Altered cofactor regulation with disease-associated p97/VCP mutations.

    PubMed

    Zhang, Xiaoyi; Gui, Lin; Zhang, Xiaoyan; Bulfer, Stacie L; Sanghez, Valentina; Wong, Daniel E; Lee, YouJin; Lehmann, Lynn; Lee, James Siho; Shih, Pei-Yin; Lin, Henry J; Iacovino, Michelina; Weihl, Conrad C; Arkin, Michelle R; Wang, Yanzhuang; Chou, Tsui-Fen

    2015-04-01

    Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis. PMID:25775548

  2. 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

  3. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex.

    PubMed

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-12-06

    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.

  4. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme.

    PubMed

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-07-21

    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 Mg(2+) 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 Mg(2+) 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.

  5. Nanotube-supported bioproduction of 4-hydroxy-2-butanone via in situ cofactor regeneration.

    PubMed

    Wang, Liang; Zhang, Hongfang; Ching, Chi-Bun; Chen, Yuan; Jiang, Rongrong

    2012-06-01

    Nicotinamide cofactor-dependent oxidoreductases have been widely employed during the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these biotransformation reactions. Herein, we report the synthesis of an important pharmaceutical intermediate 4-hydroxy-2-butanone (4H2B) via an immobilized in situ cofactor regeneration system composed of NAD(+)-dependent glycerol dehydrogenase (GlyDH) and NAD(+)-regenerating NADH oxidase (nox). Both enzymes were immobilized on functionalized single-walled carbon nanotubes (SWCNTs) through the specific interaction between the His-tagged enzymes and the modified SWCNTs. GlyDH demonstrated ca. 100% native enzyme activity after immobilization. The GlyDH/nox ratio, pH, and amount of nicotinamide cofactor were examined to establish the optimum reaction conditions for 4H2B production. The nanoparticle-supported cofactor regeneration system become more stable and the yield of 4H2B turned out to be almost twice (37%) that of the free enzyme system after a 12-h reaction. Thus, we believe that this non-covalent specific immobilization procedure can be applied to cofactor regeneration system for bioconversions.

  6. 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.

  7. 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

  8. 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 Central

    Chow, T J; Tabita, F R

    1994-01-01

    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. Images PMID:7928999

  9. Identification of Two New Genes Involved in Diazotrophic Growth via the Alternative Fe-Only Nitrogenase in the Phototrophic Purple Bacterium Rhodobacter capsulatus

    PubMed Central

    Sicking, Christa; Brusch, Margit; Lindackers, Andreas; Riedel, Kai-Uwe; Schubert, Britta; Isakovic, Nazila; Krall, Christiane; Klipp, Werner; Drepper, Thomas; Schneider, Klaus; Masepohl, Bernd

    2005-01-01

    Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed. PMID:15601692

  10. Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus.

    PubMed

    Sicking, Christa; Brusch, Margit; Lindackers, Andreas; Riedel, Kai-Uwe; Schubert, Britta; Isakovic, Nazila; Krall, Christiane; Klipp, Werner; Drepper, Thomas; Schneider, Klaus; Masepohl, Bernd

    2005-01-01

    Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed. PMID:15601692

  11. Homologous regulators, CnfR1 and CnfR2, activate expression of two distinct nitrogenase gene clusters in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

    PubMed

    Pratte, Brenda S; Thiel, Teresa

    2016-06-01

    The cyanobacterium Anabaena variabilis has two Mo-nitrogenases that function under different environmental conditions in different cell types. The heterocyst-specific nitrogenase encoded by the large nif1 gene cluster and the similar nif2 gene cluster that functions under anaerobic conditions in vegetative cells are under the control of the promoter for the first gene of each cluster, nifB1 or nifB2 respectively. Associated with each of these clusters is a putative regulatory gene called cnfR (patB) whose product has a C-terminal HTH domain and an N-terminal ferredoxin-like domain. CnfR1 activates nifB1 expression in heterocysts, while CnfR2 activates nifB2 expression. A cnfR1 mutant was unable to make nitrogenase under aerobic conditions in heterocysts while the cnfR2 mutant was unable to make nitrogenase under anaerobic conditions. Mutations in cnfR1 and cnfR2 reduced transcripts for the nif1 and nif2 genes respectively. The closely related cyanobacterium, Anabaena sp. PCC 7120 has the nif1 system but lacks nif2. Expression of nifB2:lacZ from A. variabilis in anaerobic vegetative cells of Anabaena sp. PCC 7120 depended on the presence of cnfR2. This suggests that CnfR2 is necessary and sufficient for activation of the nifB2 promoter and that the CnfR1/CnfR2 family of proteins are the primary activators of nitrogenase gene expression in cyanobacteria. PMID:26950042

  12. Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia.

    PubMed

    Richau, K H; Kudahettige, R L; Pujic, P; Kudahettige, N P; Sellstedt, A

    2013-11-01

    The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this 'waste' product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under freeliving conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The

  13. The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii.

    PubMed Central

    Joerger, R D; Wolfinger, E D; Bishop, P E

    1991-01-01

    Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium. In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion. Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels. An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo

  14. The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii.

    PubMed

    Joerger, R D; Wolfinger, E D; Bishop, P E

    1991-07-01

    Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium. In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion. Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels. An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo

  15. Sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues.

    PubMed

    Hanz, Gisela Maria; Jung, Britta; Giesbertz, Anna; Juhasz, Matyas; Weinhold, Elmar

    2014-11-22

    S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as

  16. Regulation of estrogen-dependent transcription by the LIM cofactors CLIM and RLIM in breast cancer.

    PubMed

    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 (ERalpha). Although it is known that ERalpha 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-HD) 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 ERalpha cofactors that colocalize and interact with ERalpha in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERalpha target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERalpha and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERalpha on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERalpha target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERalpha during the development of human breast cancer.

  17. 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.

  18. 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

  19. Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae.

    PubMed

    Bloem, Audrey; Sanchez, Isabelle; Dequin, Sylvie; Camarasa, Carole

    2015-10-16

    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.

  20. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.

    PubMed

    Wang, Liying; Zhang, Lihong; Liu, Zhanzhi; Liu, Zhangzhi; 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 σ(70) (σ(A))-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.

  1. 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

  2. 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.

  3. [On the influence of local molecular environment on the redox potential of electron transfer cofactors in bacterial photosynthetic reaction centers].

    PubMed

    Krasil'nikov, P M; Noks, P P; Rubin, A B

    2011-01-01

    The addition of cryosolvents (glycerol, dimethylsulfoxide) to a water solution containing bacterial photosynthetic reaction centers changes the redox potential of the bacteriochlorophyll dimer, but does not affect the redox potential of the quinone primary acceptor. It has been shown that the change in redox potential can be produced by changes of the electrostatic interactions between cofactors and the local molecular environment modified by additives entered into the solution. The degree of influence of a solvent on the redox potential of various cofactors is determined by degree of availability of these cofactors for molecules of solvent, which depends on the arrangement of cofactors in the structure of reaction centers.

  4. Computational study of the Fe(CN)2CO cofactor and its binding to HypC protein.

    PubMed

    Albareda, Marta; Palacios, Jose-Manuel; Imperial, Juan; Pacios, Luis F

    2013-10-31

    In the intricate maturation process of [NiFe]-hydrogenases, the Fe(CN)2CO cofactor is first assembled in a HypCD complex with iron coordinated by cysteines from both proteins and CO is added after ligation of cyanides. The small accessory protein HypC is known to play a role in delivering the cofactor needed for assembling the hydrogenase active site. However, the chemical nature of the Fe(CN)2CO moiety and the stability of the cofactor-HypC complex are open questions. In this work, we address geometries, properties, and the nature of bonding of all chemical species involved in formation and binding of the cofactor by means of quantum calculations. We also study the influence of environmental effects and binding to cysteines on vibrational frequencies of stretching modes of CO and CN used to detect the presence of Fe(CN)2CO. Carbon monoxide is found to be much more sensitive to sulfur binding and the polarity of the medium than cyanides. The stability of the HypC-cofactor complex is analyzed by means of molecular dynamics simulation of cofactor-free and cofactor-bound forms of HypC. The results show that HypC is stable enough to carry the cofactor, but since its binding cysteine is located at the N-terminal unstructured tail, it presents large motions in solution, which suggests the need for a guiding interaction to achieve delivery of the cofactor.

  5. 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.

  6. Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

    PubMed

    Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

    2016-02-15

    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.

  7. One diagonal texture or cofactor zero of the neutrino mass matrix

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

    In view of the recent measurement of nonzero θ13, we carry out a systematic study of a simple class of neutrino models that has one diagonal texture or cofactor zero in the mass matrix. There are seven free parameters in the model, and five of them are already measured by neutrino oscillation experiments; some cases for the normal or inverted hierarchy are excluded, and for the rest we obtain the preferred values for the lightest neutrino mass and Dirac CP phase. We find that there are strong similarities between one diagonal texture zero models with one mass hierarchy and one diagonal cofactor zero models with the opposite mass hierarchy. We also make predictions for neutrinoless double beta decay for these models. For the one cofactor zero models, we present a simple realization based on a new U(1) gauge symmetry.

  8. 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.

  9. 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.

  10. 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. PMID:26616922

  11. 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.

  12. Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

    PubMed

    Morris, P; Rios-Solis, L; García-Arrazola, R; Lye, G J; Dalby, P A

    2016-07-01

    Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

  13. 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.

  14. 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.

  15. 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. PMID:22210458

  16. 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

  17. Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors

    SciTech Connect

    Allen, J.P.; Feher, G.; Yeates, T.O.; Komiya, H.; Rees, D.C.

    1987-08-01

    The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 A with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure.

  18. 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.

  19. Evidence for pyrroloquinolinequinone as the carbonyl cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy.

    PubMed

    Williamson, P R; Moog, R S; Dooley, D M; Kagan, H M

    1986-12-15

    The present study investigated the possibility that pyrroloquinolinequinone (PQQ), an aromatic carbonyl recently indicated to be the carbonyl cofactor in bovine plasma amine oxidase, may also be present at the active site of lysyl oxidase. The absorption and resonance Raman spectra of the phenylhydrazones of bovine plasma amine oxidase, of peptides derived from the active site of bovine aorta lysyl oxidase, and of PQQ were very similar, indicating that the carbonyl cofactor of lysyl oxidase is PQQ or a compound which closely resembles PQQ.

  20. Mono and dual cofactor dependence of human cystathionine β-synthase enzyme variants in vivo and in vitro.

    PubMed

    Dimster-Denk, Dago; Tripp, Katherine W; Marini, Nicholas J; Marqusee, Susan; Rine, Jasper

    2013-10-03

    Any two individuals differ from each other by an average of 3 million single-nucleotide polymorphisms. Some polymorphisms have a functional impact on cofactor-using enzymes and therefore represent points of possible therapeutic intervention through elevated-cofactor remediation. Because most known disease-causing mutations affect protein stability, we evaluated how the in vivo impact caused by single amino acid substitutions in a prototypical enzyme of this type compared with physical characteristics of the variant enzymes in vitro. We focused on cystathionine β-synthase (CBS) because of its clinical relevance in homocysteine metabolism and because some variants of the enzyme are clinically responsive to increased levels of its B6 cofactor. Single amino-acid substitutions throughout the CBS protein caused reduced function in vivo, and a subset of these altered sensitivity to limiting B6-cofactor. Some of these B6-sensitive substitutions also had altered sensitivity to limiting heme, another CBS cofactor. Limiting heme resulted in reduced incorporation of heme into these variants, and subsequently increased protease sensitivity of the enzyme in vitro. We hypothesize that these alleles caused a modest, yet significant, destabilization of the native state of the protein, and that the functional impact of the amino acid substitutions caused by these alleles can be influenced by cofactor(s) even when the affected amino acid is distant from the cofactor binding site.

  1. Mono and Dual Cofactor Dependence of Human Cystathionine β-Synthase Enzyme Variants In Vivo and In Vitro

    PubMed Central

    Dimster-Denk, Dago; Tripp, Katherine W.; Marini, Nicholas J.; Marqusee, Susan; Rine, Jasper

    2013-01-01

    Any two individuals differ from each other by an average of 3 million single-nucleotide polymorphisms. Some polymorphisms have a functional impact on cofactor-using enzymes and therefore represent points of possible therapeutic intervention through elevated-cofactor remediation. Because most known disease-causing mutations affect protein stability, we evaluated how the in vivo impact caused by single amino acid substitutions in a prototypical enzyme of this type compared with physical characteristics of the variant enzymes in vitro. We focused on cystathionine β-synthase (CBS) because of its clinical relevance in homocysteine metabolism and because some variants of the enzyme are clinically responsive to increased levels of its B6 cofactor. Single amino-acid substitutions throughout the CBS protein caused reduced function in vivo, and a subset of these altered sensitivity to limiting B6-cofactor. Some of these B6-sensitive substitutions also had altered sensitivity to limiting heme, another CBS cofactor. Limiting heme resulted in reduced incorporation of heme into these variants, and subsequently increased protease sensitivity of the enzyme in vitro. We hypothesize that these alleles caused a modest, yet significant, destabilization of the native state of the protein, and that the functional impact of the amino acid substitutions caused by these alleles can be influenced by cofactor(s) even when the affected amino acid is distant from the cofactor binding site. PMID:23934999

  2. Structural basis of the cofactor function of denatured albumin in plasminogen activation by tissue-type plasminogen activator.

    PubMed

    Galántai, Rita; Módos, Károly; Fidy, Judit; Kolev, Krasimir; Machovich, Raymund

    2006-03-17

    Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100-230 microM albumin (80 degrees C and 60-90 min) reproducibly yielded aggregates with radius in the range of 10-150 nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10 nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular beta-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein. PMID:16438933

  3. A modular system for regeneration of NAD cofactors using graphite particles modified with hydrogenase and diaphorase moieties.

    PubMed

    Reeve, Holly A; Lauterbach, Lars; Ash, Philip A; Lenz, Oliver; Vincent, Kylie A

    2012-02-01

    Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase. PMID:21986817

  4. Cellular cofactors of lentiviral integrase: from target validation to drug discovery.

    PubMed

    Taltynov, Oliver; Desimmie, Belete A; Demeulemeester, Jonas; Christ, Frauke; Debyser, Zeger

    2012-01-01

    To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN), an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75) is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2) has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs).

  5. 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.

  6. Interactions between the estrogen receptor, its cofactors and microRNAs in breast cancer.

    PubMed

    McCafferty, Marc P J; McNeill, Roisin E; Miller, Nicola; Kerin, Michael J

    2009-08-01

    The activity of selective estrogen receptor modulators (SERMs) is not fully explained by an estrogen receptor (ER) switch model that simply turns estrogen activity on or off. A better understanding of the mechanisms involved in estrogen signaling and the development of drug resistance could help stratify patients into more coherent treatment groups and identify novel therapeutic candidates. This review describes how interactions between two novel factors known to influence estrogenic activity: nuclear receptor cofactors--protein partners which modulate estrogen action, and microRNAs--a class of recently discovered regulatory elements, may impact hormone-sensitive breast cancer. The role of nuclear receptor cofactors in estrogen signaling and the associations between ER cofactors and breast cancer are described. We outline the activity of microRNAs (miRNAs) and their associations with breast cancer and detail recent evidence of interactions between the ER and its cofactors and miRNA and provide an overview of the emerging field of miRNA-based therapeutics. We propose that previously unrecognised interactions between these two species of regulatory molecules may underlie at least some of the heterogeneity of breast cancer in terms of its clinical course and response to treatment. The exploitation of such associations will have important implications for drug development.

  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. 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.

  9. An unexpected reactivity of the P460 cofactor in hydroxylamine oxidoreductase.

    PubMed

    Dietl, Andreas; Maalcke, Wouter; Barends, Thomas R M

    2015-08-01

    Hydroxylamine oxidoreductases (HAOs) contain a unique haem cofactor called P460 that consists of a profoundly ruffled c-type haem with two covalent bonds between the haem porphyrin and a conserved tyrosine. This cofactor is exceptional in that it abstracts electrons from a ligand bound to the haem iron, whereas other haems involved in redox chemistry usually inject electrons into their ligands. The effects of the tyrosine cross-links and of the haem ruffling on the chemistry of this cofactor have been investigated theoretically but are not yet clear. A new crystal structure of an HAO from Candidatus Kuenenia stuttgartiensis, a model organism for anaerobic ammonium oxidation, now shows that its P460 cofactor has yet another unexpected reactivity: when ethylene glycol was used as a cryoprotectant, the 1.8 Å resolution electron-density maps showed additional density which could be interpreted as an ethylene glycol molecule covalently bound to the C16 atom of the haem ring, opposite the covalent links to the conserved tyrosine. Possible causes for this unexpected reactivity are discussed.

  10. Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor.

    PubMed

    Glatz, Z; Kovár, J; Macholán, L; Pec, P

    1987-03-01

    Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.

  11. RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro.

    PubMed Central

    Kretzschmar, M; Stelzer, G; Roeder, R G; Meisterernst, M

    1994-01-01

    We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II. Images PMID:8196633

  12. 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.

  13. 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

  14. Photosensitivity syndrome brings to light a new transcription-coupled DNA repair cofactor.

    PubMed

    Cleaver, James E

    2012-05-01

    Three teams have applied whole-exome and proteome methods to identify a new cofactor of human RNA polymerase II that is required for the recovery of transcription on damaged templates. The identification of this new factor raises questions about the causal relationships between molecular mechanisms of transcription regulation and excision repair and developmental and neurological disease and nonmalignant skin photosensitivity. PMID:22538718

  15. 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.

  16. Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways.

    PubMed

    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

    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

  17. 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. PMID:27164259

  18. 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.

  19. How Does Protein Architecture Facilitate the Transduction of ATP Chemical-Bond Energy into Mechanical Work? The Cases of Nitrogenase and ATP Binding-Cassette Proteins

    PubMed Central

    Liao, Jie-Lou; Beratan, David N.

    2004-01-01

    Transduction of adenosine triphosphate (ATP) chemical-bond energy into work to drive large-scale conformational changes is common in proteins. Two specific examples of ATP-utilizing proteins are the nitrogenase iron protein and the ATP binding-cassette transporter protein, BtuCD. Nitrogenase catalyzes biological nitrogen fixation whereas BtuCD transports vitamin B12 across membranes. Both proteins drive their reactions with ATP. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated in these proteins, a coarse-grained elastic network model is employed. The analysis shows that subunits of the proteins move against each other in a concerted manner. The lowest-frequency modes of the nitrogenase iron protein and of the ATP binding-cassette transporter BtuCD protein are found to link the functionally critical domains, and these modes are suggested to be responsible for (at least the initial stages) large-scale ATP-coupled conformational changes. PMID:15298939

  20. Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness.

    PubMed Central

    Li, J D; Hu, C Z; Yoch, D C

    1987-01-01

    Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+- or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool remained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4+ azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4+ substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivation. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4+ switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4+ switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4+ (glutamine) switch-off. Images PMID:2878918

  1. A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

    PubMed

    Ronau, Judith A; Paul, Lake N; Fuchs, Julian E; Liedl, Klaus R; Abu-Omar, Mahdi M; Das, Chittaranjan

    2014-11-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  2. Genomewide markers as cofactors for precision mapping of quantitative trait loci.

    PubMed

    Bernardo, R

    2013-04-01

    In composite interval mapping of quantitative trait loci (QTL), subsets of background markers are used to account for the effects of QTL outside the marker interval being tested. Here, I propose a QTL mapping approach (called G model) that utilizes genomewide markers as cofactors. The G model involves backward elimination on a given chromosome after correcting for genomewide marker effects, calculated under a random effects model, at all the other chromosomes. I simulated a trait controlled by 15 or 30 QTL, mapping populations of N = 96, 192, and 384 recombinant inbreds, and N M = 192 and 384 evenly spaced markers. In the C model, which utilized subsets of background markers, the number of QTL detected and the number of false positives depended on the number of cofactors used, with five cofactors being too few with N = 384 and 20-40 cofactors being too many with N = 96. A window size of 0 cM for excluding cofactors maintained the number of true QTL detected while decreasing the number of false positives. The number of true QTL detected was generally higher with the G model than with the C model, and the G model led to good control of the type I error rate in simulations where the null hypothesis of no marker-QTL linkage was true. Overall, the results indicated that the G model is useful in QTL mapping because it is less subjective and has equal, if not better, performance when compared with the traditional approach of using subsets of markers to account for background QTL.

  3. 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.

  4. Mutations of an NAD(P)H-dependent flavoprotein monooxygenase that influence cofactor promiscuity and enantioselectivity.

    PubMed

    Jensen, Chantel N; Ali, Sohail T; Allen, Michael J; Grogan, Gideon

    2013-01-01

    The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2'-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(k cat/K m NADH)/k cat/K m NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced K m for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e., compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.

  5. 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

  6. Mechanisms of the S/CO/Se interchange reactions at FeMo-co, the active site cluster of nitrogenase.

    PubMed

    Dance, Ian

    2016-09-28

    The active site of the N2 fixing enzyme nitrogenase is a C-centred Fe7MoS cluster (FeMo-co) containing a trigonal prism of six Fe atoms connected by a central belt of three doubly-bridging S atoms. The trigonal faces of the prism are capped via triply-bridging S atoms to Fe1 at one end and Mo at the other end. One of the central belt atoms, S2B, considered to be important in the chemical mechanism of the enzyme, has been shown by Spatzal, Rees et al. to undergo substitution by CO, and also substitution by Se in the presence of SeCN(-), under turnover conditions. Further, when turning over under C2H2 or N2/CO there is migration of Se to the other two belt bridging positions. These reactions are extraordinary, and unprecedented in metal chalcogenide cluster chemistry. Using density functional simulations, mechanisms for all of these reactions have been developed, involving the small molecules SCO, SeCO, C2H2S, C2H2Se, SeCN(-), SCN(-) functioning as carriers of S and Se atoms. The possibility that the S2B bridge position is vacant is discounted, because the barrier to formation of a bridge-void intermediate with two contiguous three-coordinate Fe atoms is too large. A bridging ligand is retained throughout the proposed mechanisms. Intermediates with Fe-C(O)-S/Se-Fe cycles and with SCO/SeCO C-bound to Fe are predicted. The energetics of the reaction trajectories show them to be feasible and easily reversible, consistent with experiment. Alternative mechanisms involving intramolecular differential rotatory rearrangements of the cluster to scramble the Se bridges are also examined, and shown to be very unlikely. The implications of these new facets of the reactivity of the FeMo-co cluster are discussed: it is considered that they are unlikely to be part of the mechanism of the physiological reactions of nitrogenase. PMID:27534727

  7. Active sites without restraints: high-resolution analysis of metal cofactors.

    PubMed

    Burger, Eva-Maria; Andrade, Susana L A; Einsle, Oliver

    2015-12-01

    For most three-dimensional structures of biological macromolecules, the factual accuracy of atom positions by far exceeds the resolution of the experimental data, although the refinement problem presented by a protein structure is substantially underdetermined. This is achieved through using restraints that precisely define protein geometries and thus reduce the degrees of freedom of the refinement problem. If such information is not available or when unusual geometries or particular ligand states complicate structural analysis, possible pitfalls arise that not only concern the precise definition of spatial arrangements, but also the identification of atom types and bond distances. Prominent examples include CO dehydrogenase, hydrogenase, acetylene hydratase and nitrogenase, all of which employ unique active sites that turned out not to be what they seemed upon first inspection.

  8. 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

  9. 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

  10. 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.

  11. Diversity of nitrogenase (nifH) genes pool in soybean field soil after continuous and rotational cropping.

    PubMed

    Xiao, C H; Tang, H; Pu, L J; Sun, D M; Ma, J Z; Yu, M; Duan, R S

    2010-08-01

    Diazotrophs diversity in soybean is a topic requiring thorough investigation since the previous researches have focused on only rice, forest, grass, water, etc. In this research, iron-only nitrogenase nifH gene was as genetic marker. PCR-RFLP was used to investigate the difference of diazotrophs community diversity in the soil from the continuous cropping (CC) (the 5-yr tilling of soybean) and the rotational cropping (RC) (soybean-corn) soils in the northeast of China. A total of 36 isolates were genetically characterized. Most of the isolates closely related to Azospirillum and Azotobacter. Eighty-six unique nifH gene sequences were obtained by cloning of the respective PCR products in two soil samples. It was found that the diversity of nifH genes in CC changed obviously compared with RC. Phylogenetic analysis indicated that most of the clones clustered together in a high homogeneity with some sequence retrieved from environmental representatives. The sequence diversity of nifH genes was high and the members of the Alphaproteobacteria were predominant in both samples. The experimental study also revealed the two non-proteobacterial diazotrophs, firmicutes and euryarchaeota. Through this study, it can be assumed that different tillage perhaps affected the nifH gene-containing population diversity.

  12. 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.

  13. 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.

  14. 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 Central

    2015-01-01

    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 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. PMID:25275608

  15. 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.

  16. ChIPModule: systematic discovery of transcription factors and their cofactors from ChIP-seq data.

    PubMed

    Ding, Jun; Cai, Xiaohui; Wang, Ying; Hu, Haiyan; Li, Xiaoman

    2013-01-01

    We have developed a novel approach called ChIPModule to systematically discover transcription factors and their cofactors from ChIP-seq data. Given a ChIP-seq dataset and the binding patterns of a large number of transcription factors, ChIPModule can efficiently identify groups of transcription factors, whose binding sites significantly co-occur in the ChIP-seq peak regions. By testing ChIPModule on simulated data and experimental data, we have shown that ChIPModule identifies known cofactors of transcription factors, and predicts new cofactors that are supported by literature. ChIPModule provides a useful tool for studying gene transcriptional regulation.

  17. Selective detection of NADPH among four pyridine-nucleotide cofactors by a fluorescent probe based on aggregation-induced emission.

    PubMed

    Noguchi, Takao; Dawn, Arnab; Yoshihara, Daisuke; Tsuchiya, Youichi; Yamamoto, Tatsuhiro; Shinkai, Seiji

    2013-05-14

    A fluorescent sensor based on guanidinium-tethered tetraphenylethene (TPE) has been investigated toward the differentiation of pyridine nucleotide cofactors (NAD(+) , NADH, NADP(+) , and NADPH). TPE selectively recognizes NADPH possessing the higher tetra-anionic net-charge, resulting in the steep "turn-on" fluorescence increase. The comparative aggregation behaviors and fluorescence response studies of TPE on the four cofactors reveal that the critical aggregate concentration of TPE against NADPH correlates directly with the concentration threshold for the fluorescence response. These results establish that TPE can selectively differentiate NADPH over the other three cofactors by the steep aggregation-induced fluorescence response accompanied by the high signal-to-background contrast.

  18. 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

  19. Relationship between methanogenic cofactor content and maximum specific methanogenic activity of anaerobic granular sludges

    SciTech Connect

    Gorris, L.G.; de Kok, T.M.; Kroon, B.M.; van der Drift, C.; Vogels, G.D.

    1988-05-01

    In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F/sub 420/-2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F/sub 420/-4 and F/sub 420/-5 sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.

  20. Role of HOXA9 in leukemia: dysregulation, cofactors and essential targets.

    PubMed

    Collins, C T; Hess, J L

    2016-03-01

    HOXA9 is a homeodomain-containing transcription factor that has an important role in hematopoietic stem cell expansion and is commonly deregulated in acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia lead to overexpression of HOXA9, which is a strong predictor of poor prognosis. In many cases, HOXA9 has been shown to be necessary for maintaining leukemic transformation; however, the molecular mechanisms through which it promotes leukemogenesis remain elusive. Recent work has established that HOXA9 regulates downstream gene expression through binding at promoter distal enhancers along with a subset of cell-specific cofactor and collaborator proteins. Increasing efforts are being made to identify both the critical cofactors and target genes required for maintaining transformation in HOXA9-overexpressing leukemias. With continued advances in understanding HOXA9-mediated transformation, there is a wealth of opportunity for developing novel therapeutics that would be applicable for greater than 50% of AML with overexpression of HOXA9.

  1. 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

  2. Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase

    SciTech Connect

    Tu, Chao; Tropea, Joseph E.; Austin, Brian P.; Court, Donald L.; Waugh, David S.; Ji, Xinhua

    2009-03-27

    Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs IVIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.

  3. Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

    PubMed

    Groen, B W; van der Meer, R A; Duine, J A

    1988-09-12

    Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

  4. Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei.

    PubMed

    Djikeng, Appolinaire; Raverdy, Sylvine; Foster, Jeremy; Bartholomeu, Daniella; Zhang, Yinhua; El-Sayed, Najib M; Carlow, Clotilde

    2007-03-01

    Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.

  5. Human GLI-2 Is a Tat Activation Response Element-Independent Tat Cofactor

    PubMed Central

    Browning, Catherine M.; Smith, Michael J.; Clark, Nina M.; Lane, Brian R.; Parada, Camilo; Montano, Monty; KewalRamani, Vineet N.; Littman, Dan R.; Essex, Max; Roeder, Robert G.; Markovitz, David M.

    2001-01-01

    Zinc finger-containing GLI proteins are involved in the development of Caenorhabditis elegans, Xenopus, Drosophila, zebrafish, mice, and humans. In this study, we show that an isoform of human GLI-2 strongly synergizes with the Tat transactivating proteins of human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and markedly stimulates viral replication. GLI-2 also synergizes with the previously described Tat cofactor cyclin T1 to stimulate Tat function. Surprisingly, GLI-2/Tat synergy is not dependent on either a typical GLI DNA binding site or an intact Tat activation response element but does require an intact TATA box. Thus, GLI-2/Tat synergy results from a mechanism of action which is novel both for a GLI protein and for a Tat cofactor. These findings link the GLI family of transcriptional and developmental regulatory proteins to Tat function and HIV replication. PMID:11160734

  6. A Functional Genomic Screen Identifies Cellular Cofactors of Hepatitis C Virus Replication

    PubMed Central

    Tai, Andrew W.; Benita, Yair; Peng, Lee F.; Kim, Sun-Suk; Sakamoto, Naoya; Xavier, Ramnik J.; Chung, Raymond T.

    2009-01-01

    SUMMARY Hepatitis C virus (HCV) chronically infects 3% of the world’s population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies. PMID:19286138

  7. CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site.

    PubMed

    Kwak, Yeonju; Schwartz, Jennifer K; Huang, Victor W; Boice, Emily; Kurtz, Donald M; Solomon, Edward I

    2015-12-01

    Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

  8. Biosynthesis of flavin cofactors in man: implications in health and disease.

    PubMed

    Barile, Maria; Giancaspero, Teresa Anna; Brizio, Carmen; Panebianco, Concetta; Indiveri, Cesare; Galluccio, Michele; Vergani, Lodovica; Eberini, Ivano; Gianazza, Elisabetta

    2013-01-01

    The primary role of the water-soluble vitamin B2, i.e. riboflavin, in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding as well as in diverse regulatory events. Deficiency of riboflavin in men and experimental animal models has been linked to several diseases, including neuromuscular and neurological disorders and cancer. Riboflavin at pharmacological doses has been shown to play unexpected and incompletely understood regulatory roles. Besides a summary on riboflavin uptake and a survey on riboflavin-related diseases, the main focus of this review is on discovery and characterization of FAD synthase (EC 2.7.7.2) and other components of the cellular networks that ensure flavin cofactor homeostasis.Special attention is devoted to the problem of sub-cellular compartmentalization of cofactor synthesis in eukaryotes, made possible by the existence of different FAD synthase isoforms and specific molecular components involved in flavin trafficking across sub-cellular membranes.Another point addressed in this review is the mechanism of cofactor delivery to nascent apo-proteins, especially those localized into mitochondria, where they integrate FAD in a process that involves additional mitochondrial protein(s) still to be identified. Further efforts are necessary to elucidate the role of riboflavin/FAD network in human pathologies and to exploit the structural differences between human and microbial/fungal FAD synthase as the rational basis for developing novel antibiotic/antimycotic drugs. PMID:23116402

  9. 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.

  10. Evidence for methoxatin (pyrroloquinolinequinone) as the cofactor in bovine plasma amine oxidase from resonance Raman spectroscopy.

    PubMed Central

    Moog, R S; McGuirl, M A; Cote, C E; Dooley, D M

    1986-01-01

    Resonance Raman spectra of the 2,4-dinitrophenylhydrazine derivatives of bovine plasma amine oxidase [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] have been measured. Detailed comparisons to the spectra of the corresponding derivatives of methoxatin (pyrroloquinolinequinone), pyridoxal, and other aldehydes and diones provide further evidence that covalently bound methoxatin or a closely similar derivative is the organic cofactor in copper-containing amine oxidases. PMID:3464962

  11. Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo†

    PubMed Central

    Cotruvo, Joseph A.; Stubbe, JoAnne

    2011-01-01

    Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (FeIII2-Y•) and dimanganese(III)-Y• (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo J.A., Jr. and Stubbe J., Biochemistry (2010) 49, 1297–1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ~95% purity, with 0.2 Y•/β2, 0.9 Mn/β2, and a specific activity of 720 nmol/min/mg. In these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo. PMID:21250660

  12. DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation.

    PubMed

    Prochazkova, Jana; Sakaguchi, Shinya; Owusu, Michel; Mazouzi, Abdelghani; Wiedner, Marc; Velimezi, Georgia; Moder, Martin; Turchinovich, Gleb; Hladik, Anastasiya; Gurnhofer, Elisabeth; Hayday, Adrian; Behrens, Axel; Knapp, Sylvia; Kenner, Lukas; Ellmeier, Wilfried; Loizou, Joanna I

    2015-11-01

    Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.

  13. Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment.

    PubMed

    Woodham, Andrew W; Skeate, Joseph G; Sanna, Adriana M; Taylor, Julia R; Da Silva, Diane M; Cannon, Paula M; Kast, W Martin

    2016-07-01

    In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.

  14. 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.

  15. Quantum localization and protein-assisted vibrational energy flow in cofactors

    NASA Astrophysics Data System (ADS)

    Leitner, David M.

    2010-08-01

    Quantum effects influence vibrational dynamics and energy flow in biomolecules, which play a central role in biomolecule function, including control of reaction kinetics. Lifetimes of many vibrational modes of proteins and their temperature dependence, as determined by quantum golden-rule-based calculations, exhibit trends consistent with experimental observation and distinct from estimates based on classical modeling. Particularly notable are quantum coherence effects that give rise to localization of vibrational states of sizable organic molecules in the gas phase. Even when such a molecule, for instance a cofactor, is embedded in a protein, remnants of quantum localization survive that influence vibrational energy flow and its dependence on temperature. We discuss these effects on the mode-damping rates of a cofactor embedded in a protein, using the green fluorescent protein chromophore as a specific example. We find that for cofactors of this size embedded in their protein and solvent environment at room temperature a golden-rule calculation often overestimates the mode-damping rate.

  16. Regulation of the human Suv3 helicase on DNA by inorganic cofactors.

    PubMed

    Venø, Susanne T; Witt, Marie B; Kulikowicz, Tomasz; Bohr, Vilhelm A; Stevnsner, Tinna

    2015-01-01

    Mitochondria are essential organelles and consequently proper expression and maintenance of the mitochondrial genome are indispensable for proper cell function. The mitochondrial Suv3 (SUPV3L1) helicase is known to have a central role in mitochondrial RNA metabolism and to be essential for maintenance of mitochondrial DNA stability. Here we have performed biochemical investigations to determine the potential regulation of the human Suv3 (hSuv3) helicase function by inorganic cofactors. We find that hSuv3 helicase and ATPase activity in vitro is strictly dependent on the presence of specific divalent cations. Interestingly, we show that divalent cations and nucleotide concentration have a direct effect on helicase substrate stability. Also, hSuv3 helicase is able to utilize several different nucleotide cofactors including both NTPs and dNTPs. Intriguingly, the potency of the individual nucleotide as energy source for hSuv3 unwinding differed depending on the included divalent cation and nucleotide concentration. At low concentrations, all four NTPs could support helicase activity with varying effectiveness depending on the included divalent cation. However, at higher nucleotide concentrations, only ATP was able to elicit the helicase activity of hSuv3. Consequently, we speculate that the capacity of hSuv3 DNA unwinding activity might be sensitive to the local availability of specific inorganic cofactors.

  17. Metal cofactors in the structure and activity of the fowlpox resolvase.

    PubMed

    Culyba, Matthew J; Hwang, Young; Hu, Jimmy Yan; Minkah, Nana; Ocwieja, Karen E; Bushman, Frederic D

    2010-05-28

    Poxvirus DNA replication generates linear concatemers containing many copies of the viral genome with inverted repeat sequences at the junctions between monomers. The inverted repeats refold to generate Holliday junctions, which are cleaved by the virus-encoded resolvase enzyme to form unit-length genomes. Here we report studies of the influence of metal cofactors on the activity and structure of the resolvase of fowlpox virus, which provides a tractable model for in vitro studies. Small-molecule inhibitors of related enzymes bind simultaneously to metal cofactors and nearby surface amino acid residues, so understanding enzyme-cofactor interactions is important for the design of antiviral agents. Analysis of inferred active-site residues (D7, E60, K102, D132, and D135) by mutagenesis and metal rescue experiments specified residues that contribute to binding metal ions and that multiple binding sites are probably involved. Differential electrophoretic analysis was used to map the conformation of the DNA junction when bound by resolvase. For the wild-type complex in the presence of EDTA (ethylenediaminetetraacetic acid) or Ca(2+), migration was consistent with the DNA arms arranged in near-tetrahedral geometry. However, the D7N active-site mutant resolvase held the arms in a more planar arrangement in EDTA, Ca(2+), or Mg(2+) conditions, implicating metal-dependent contacts at the active site in the larger architecture of the complex. These data show how divalent metals dictate the conformation of FPV resolvase-DNA complexes and subsequent DNA cleavage.

  18. Intracellular trafficking of the pyridoxal cofactor. Implications for health and metabolic disease.

    PubMed

    Whittaker, James W

    2016-02-15

    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.

  19. 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.

  20. 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.

  1. 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.

  2. The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria *

    PubMed Central

    Whittaker, Mei M.; Penmatsa, Aravind; Whittaker, James W.

    2015-01-01

    Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5′-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial ‘PLP-ome’) was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1+) and knockout (MTM1−) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration. PMID:25637770

  3. 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

  4. DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation

    PubMed Central

    Prochazkova, Jana; Sakaguchi, Shinya; Owusu, Michel; Mazouzi, Abdelghani; Wiedner, Marc; Velimezi, Georgia; Moder, Martin; Turchinovich, Gleb; Hladik, Anastasiya; Gurnhofer, Elisabeth; Hayday, Adrian; Behrens, Axel; Knapp, Sylvia; Kenner, Lukas; Ellmeier, Wilfried; Loizou, Joanna I.

    2015-01-01

    Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1. PMID:26544571

  5. Mutagenesis studies toward understanding the mechanism of the cofactor function of thrombomodulin.

    PubMed

    Rezaie, Alireza R; Yang, Likui

    2005-10-01

    Thrombomodulin (TM) is as essential cofactor in protein C activation by thrombin. To investigate the cofactor effect of TM on the P3-P3' binding specificity of thrombin, we prepared a Gla-domainless protein C (GDPC) and an antithrombin (AT) mutant in which the P3-P3' residues of both molecules were replaced with the corresponding residues of the factor Xa cleavage site in prethrombin-2. TM is known to interact with GDPC, but not AT in the complex. Thrombin did not react with either mutant in the absence of a cofactor. While the thrombin-TM complex also did not react with the AT mutant, it activated the GDPC mutant with a normal k(cat), but an approximately 4-fold impaired K(m) value. Further studies revealed that the active-site directed inhibitor p-aminobenzamidine acts as a competitive inhibitor of both wild-type and GDPC mutant in reaction with the thrombin-TM complex. These results suggest that the interaction of the P3-P3' residues of GDPC with the active-site pocket of the thrombin-TM complex makes a dominant contribution to the binding specificity of the reaction. Moreover, the observation that the GDPC mutant, but not the AT mutant, functions as an effective substrate for the thrombin-TM complex suggests that GDPC interaction with the thrombin-TM complex may be associated with the alteration of the conformation of the P3-P3' residues of the substrate. PMID:15970373

  6. 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. PMID:27432985

  7. 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

  8. Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability.

    PubMed

    Gao, Hui; Khera, Eshita; Lee, Jung-Kul; Wen, Fei

    2016-04-22

    We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

  9. Photo-cycle dynamics of LOV1-His domain of phototropin from Chlamydomonas reinhardtii with roseoflavin monophosphate cofactor

    NASA Astrophysics Data System (ADS)

    Tyagi, A.; Penzkofer, A.; Mathes, T.; Hegemann, P.

    2010-09-01

    The wild-type phototropin protein phot from the green alga Chlamydomonas reinhardtii consists of two N-terminal LOV domains LOV1 and LOV2 with flavin mononucleotide (FMN) cofactor and a C-terminal serine-threonine kinase domain. It controls multiple steps in the sexual lifecycle of the alga. Here the LOV1-His domain of phot with modified cofactor is studied. FMN is replaced by roseoflavin monophosphate (8-dimethylamino-8-demethyl-FMN, RoFMN). The modified LOV1 domain is called RoLOV1. The photo-dynamics consequences of the cofactor change are studied. The absorption, emission, and photo-cyclic behaviour of LOV1-His and RoLOV1-His are compared. A spectroscopic characterisation of the cofactors FMN and RoFMN (roseoflavin) is given.

  10. Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae.

    PubMed

    King, Zachary A; Feist, Adam M

    2014-07-01

    Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity "swaps" for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast--including L-aspartate, L-lysine, L-isoleucine, L-proline, L-serine, and putrescine--and non-native products in E. coli-including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.

  11. Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?

    PubMed Central

    Lowe, D J; Fisher, K; Thorneley, R N

    1993-01-01

    The pre-steady-state absorbance changes that occur during the first 0.6 s of reaction of the nitrogenase of Klebsiella pneumoniae can be simulated by associating redox changes with the different states of the MoFe protein described by our published kinetic model for nitrogenase [Lowe and Thorneley (1984) Biochem. J. 224, 877-886]. When the substrate is changed, from H+ to C2H2 (acetylene) or N2, or the nitrogenase component protein ratio is altered, these pre-steady-state absorbance changes are affected in a manner that is quantitatively predicted by our model. The results, together with parallel e.p.r. studies, are interpreted as showing that the P-clusters become oxidized when the MoFe protein is in the state where bound N2 is irreversibly committed to being reduced and is protonated to the hydrazido(2-) level. PMID:8389132

  12. Reversible Photoinduced Reductive Elimination of H2 from the Nitrogenase Dihydride State, the E(4)(4H) Janus Intermediate.

    PubMed

    Lukoyanov, Dmitriy; Khadka, Nimesh; Yang, Zhi-Yong; Dean, Dennis R; Seefeldt, Lance C; Hoffman, Brian M

    2016-02-01

    We recently demonstrated that N2 reduction by nitrogenase involves the obligatory release of one H2 per N2 reduced. These studies focus on the E4(4H) "Janus intermediate", which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. E4(4H) is poised to bind and reduce N2 through reductive elimination (re) of the two hydrides as H2, coupled to the binding/reduction of N2. To obtain atomic-level details of the re activation process, we carried out in situ 450 nm photolysis of E4(4H) in an EPR cavity at temperatures below 20 K. ENDOR and EPR measurements show that photolysis generates a new FeMo-co state, denoted E4(2H)*, through the photoinduced re of the two bridging hydrides of E4(4H) as H2. During cryoannealing at temperatures above 175 K, E4(2H)* reverts to E4(4H) through the oxidative addition (oa) of the H2. The photolysis quantum yield is temperature invariant at liquid helium temperatures and shows a rather large kinetic isotope effect, KIE = 10. These observations imply that photoinduced release of H2 involves a barrier to the combination of the two nascent H atoms, in contrast to a barrierless process for monometallic inorganic complexes, and further suggest that H2 formation involves nuclear tunneling through that barrier. The oa recombination of E4(2H)* with the liberated H2 offers compelling evidence for the Janus intermediate as the point at which H2 is necessarily lost during N2 reduction; this mechanistically coupled loss must be gated by N2 addition that drives the re/oa equilibrium toward reductive elimination of H2 with N2 binding/reduction.

  13. Genes for the dimerization cofactor of hepatocyte nuclear factor-1[alpha] (DCOH) are on human and murine chromsomes 10

    SciTech Connect

    Milatovich, A.; Mendel, D.B.; Crabtree, G.R.; Francke, U. )

    1993-04-01

    Hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]; gene symbol, TCF1) forms dimers with itself as well as with HNF-1[beta] and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1[alpha], called DCOH, has been identified. Here, the authors report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids. 25 refs., 1 fig., 2 tabs.

  14. A Model of the Regulation of Nitrogenase Electron Allocation in Legume Nodules (II. Comparison of Empirical and Theoretical Studies in Soybean).

    PubMed Central

    Moloney, A. H.; Guy, R. D.; Layzell, D. B.

    1994-01-01

    In N2-fixing legumes, the proportion of total electron flow through nitrogenase (total nitrogenase activity, TNA) that is used for N2 fixation is called the electron allocation coefficient (EAC). Previous studies have proposed that EAC is regulated by the competitive inhibition of H2 on N2 fixation and that the degree of H2 inhibition can be affected by a nodule's permeability to gas diffusion. To test this hypothesis, EAC was measured in soybean (Glycine max L. Merr.) nodules exposed to various partial pressures of H2 and N2, with or without changes in TNA or nodule permeability to gas diffusion, and the results were compared with the predictions of a mathematical model that combined equations for gas diffusion and competitive inhibition of N2 fixation (A. Moloney and D.B. Layzell [1993] Plant Physiol 103: 421-428). The empirical data clearly showed that decreases in EAC were associated with increases in external pH2, decreases in external pN2, and decreases in nodule permeability to O2 diffusion. The model predicted similar trends in EAC, and the small deviations that occurred between measured and predicted values could be readily accounted for by altering one or more of the following model assumptions: K1(H2) of nitrogenase (range from 2-4% H2), Km(N2) of nitrogenase (range from 4-5% N2), the allocation of less than 100% of whole-nodule respiration to tissues within the diffusion barrier, and the presence of a diffusion pathway that is open pore versus closed pore. The differences in the open-pore and closed-pore versions of the model suggest that it may be possible to use EAC measurements as a tool for the study of legume nodule diffusion barrier structure and function. The ability of the model to predict EAC provided strong support for the hypothesis that H2 inhibition of N2 fixation plays a major role in the in vivo control of EAC and that the presence of a variable barrier to gas diffusion affects the H2 and N2 concentration in the infected cell and

  15. 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

  16. Rational proteomics II: electrostatic nature of cofactor preference in the short-chain oxidoreductase (SCOR) enzyme family.

    PubMed

    Pletnev, Vladimir Z; Weeks, Charles M; Duax, William L

    2004-11-01

    The dominant role of long-range electrostatic interatomic interactions in nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD/NADP) cofactor recognition has been shown for enzymes of the short-chain oxidoreductase (SCOR) family. An estimation of cofactor preference based only on the contribution of the electrostatic energy term to the total energy of enzyme-cofactor interaction has been tested for approximately 40 known three-dimensional (3D) crystal complexes and approximately 330 SCOR enzymes, with cofactor preference predicted by the presence of Asp or Arg recognition residues at specific 3D positions in the beta2alpha3 loop (Duax et al., Proteins 2003;53:931-943). The results obtained were found to be consistent with approximately 90% reliable cofactor assignments for those subsets. The procedure was then applied to approximately 170 SCOR enzymes with completely uncertain NAD/NADP dependence, due to the lack of Asp and Arg marker residues. The proposed 3D electrostatic approach for cofactor assignment ("3D_DeltaE(el)") has been implemented in an automatic screening procedure, and together with the use of marker residues proposed earlier (Duax et al., Proteins 2003;53:931-943), increases the level of reliable predictions for the putative SCORs from approximately 70% to approximately 90%. It is expected to be applicable for any NAD/NADP-dependent enzyme subset having at least 25-30% sequence identity, with at least one enzyme of known 3D crystal structure.

  17. 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.

  18. Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue.

    PubMed

    Ahnström, Josefin; Andersson, Helena M; Canis, Kevin; Norstrøm, Eva; Yu, Yao; Dahlbäck, Björn; Panico, Maria; Morris, Howard R; Crawley, James T B; Lane, David A

    2011-06-16

    Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.

  19. Structural and biochemical characterization of an atypical short-chain dehydrogenase/reductase reveals an unusual cofactor preference.

    PubMed

    Buysschaert, Géraldine; Verstraete, Kenneth; Savvides, Savvas N; Vergauwen, Bjorn

    2013-03-01

    Short-chain dehydrogenases/reductases (SDRs) encompass a large and functionally diverse family of enzymes with representative members in all kingdoms of life. Despite the wealth of reactions catalyzed by SDRs, they operate through a well-conserved and efficient reaction mechanism centered in a conserved catalytic tetrad (Asn-Ser-Tyr-Lys) and the employment of an appropriate cofactor. In recent years, SDRs that lack the signature catalytic tetrad have been identified, thus adding a perplexing twist to SDR functionality. In the present study, we report the crystal structure of SDRvv, an atypical SDR from Vibrio vulnificus devoid of the catalytic tetrad, thereby defining the structural signature of this apparent SDR family outlier. Further structural analysis of SDRvv in complex with its putative cofactor NADPH, site-directed mutagenesis and binding studies via isothermal titration calorimetry, and further biochemical characterization have allowed us to dissect the cofactor preferences of SDRvv. The retained capacity to bind the NADPH cofactor, the conceivable existence of a proton relay and the conservation of the coordination distances between the key residues in the cofactor binding pocket define a first set of rules towards catalytic activity for SDRvv. The findings of the present study set the stage for deriving the identity of the natural substrate of SDRvv and add a new twist to the structure-function landscape for Rossmann-fold-dependent cofactor discrimination.

  20. 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. PMID:26441891

  1. 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.

  2. Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors.

    PubMed

    Warren, Kylie; Wei, Ting; Li, Dongsheng; Qin, Fangyun; Warrilow, David; Lin, Min-Hsuan; Sivakumaran, Haran; Apolloni, Ann; Abbott, Catherine M; Jones, Alun; Anderson, Jenny L; Harrich, David

    2012-06-12

    Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

  3. Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

    SciTech Connect

    Islam, Kabirul; Chen, Yuling; Wu, Hong; Bothwell, Ian R.; Blum, Gil J.; Zeng, Hong; Dong, Aiping; Zheng, Weihong; Min, Jinrong; Deng, Haiteng; Luo, Minkui

    2013-11-18

    Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

  4. Magnetic field intensified bi-enzyme system with in situ cofactor regeneration supported by magnetic nanoparticles.

    PubMed

    Zheng, Muqing; Su, Zhiguo; Ji, Xiaoyuan; Ma, Guanghui; Wang, Ping; Zhang, Songping

    2013-10-20

    Efficient dynamic interactions among cofactor, enzymes and substrate molecules are of primary importance for multi-step enzymatic reactions with in situ cofactor regeneration. Here we showed for the first time that the above dynamic interactions could be significantly intensified by exerting an external alternating magnetic field on magnetic nanoparticles-supported multi-enzymatic system so that the inter-particle collisions due to Brownian motion of nanoparticles could be improved. To that end, a multienzyme system including glutamate dehydrogenase (GluDH), glucose dehydrogenase (GDH) and cofactor NAD(H) were separately immobilized on silica coated Fe3O4 magnetic nanoparticles with an average diameter of 105 nm, and the effect of magnetic field strength and frequency on the kinetics of the coupled bi-enzyme reaction was investigated. It was found that at low magnetic field frequency (25 Hz and 100 Hz), increasing magnetic field strength from 9.8 to 161.1 Gs led to only very slight increase in reaction rate of the coupled bi-enzyme reaction expressed by glucose consumption rate. At higher magnetic field of 200 Hz and 500 Hz, reaction rate increased significantly with increase of magnetic field strength. When the magnetic field frequency was kept at 500 Hz, the reaction rate increased from 3.89 μM/min to 8.11 μM/min by increasing magnetic field strength from 1.3 to 14.2 Gs. The immobilized bi-enzyme system also showed good reusability and stability in the magnetic field (500 Hz, 14.2 Gs), that about 46% of original activity could be retained after 33 repeated uses, accounting for totally 34 days continuous operation. These results demonstrated the feasibility in intensifying molecular interactions among magnetic nanoparticle-supported multienzymes by using nano-magnetic stirrer for efficient multi-step transformations.

  5. Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.

    PubMed Central

    Blankenfeldt, W.; Nowicki, C.; Montemartini-Kalisz, M.; Kalisz, H. M.; Hecht, H. J.

    1999-01-01

    The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group. PMID:10595543

  6. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

  7. Evidence that a Metabolic Microcompartment Contains and Recycles Private Cofactor Pools

    PubMed Central

    Huseby, Douglas L.

    2013-01-01

    Microcompartments are loose protein cages that encapsulate enzymes for particular bacterial metabolic pathways. These structures are thought to retain and perhaps concentrate pools of small, uncharged intermediates that would otherwise diffuse from the cell. In Salmonella enterica, a microcompartment encloses enzymes for ethanolamine catabolism. The cage has been thought to retain the volatile intermediate acetaldehyde but allow diffusion of the much larger cofactors NAD and coenzyme A (CoA). Genetic tests support an alternative idea that the microcompartment contains and recycles private pools of the large cofactors NAD and CoA. Two central enzymes convert ethanolamine to acetaldehyde (EutBC) and then to acetyl-CoA (EutE). Two seemingly peripheral redundant enzymes encoded by the eut operon proved to be essential for ethanolamine utilization, when subjected to sufficiently stringent tests. These are EutD (acetyl-CoA to acetyl phosphate) and EutG (acetaldehyde to ethanol). Obligatory recycling of cofactors couples the three reactions and drives acetaldehyde consumption. Loss and toxic effects of acetaldehyde are minimized by accelerating its consumption. In a eutD mutant, acetyl-CoA cannot escape the compartment but is released by mutations that disrupt the structure. The model predicts that EutBC (ethanolamine-ammonia lyase) lies outside the compartment, using external coenzyme B12 and injecting its product, acetaldehyde, into the lumen, where it is degraded by the EutE, EutD, and EutG enzymes using private pools of CoA and NAD. The compartment appears to allow free diffusion of the intermediates ethanol and acetyl-PO4 but (to our great surprise) restricts diffusion of acetaldehyde. PMID:23585538

  8. Non-racemic Antifolates Stereo-selectively Recruit Alternate Cofactors and Overcome Resistance in S. aureus

    PubMed Central

    Keshipeddy, Santosh; Reeve, Stephanie M.; Anderson, Amy C.; Wright, Dennis L.

    2016-01-01

    While antifolates such as Bactrim (trimethoprim-sulfamethoxazole; TMP-SMX) continue to play an important role in treating community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), resistance-conferring mutations, specifically F98Y of dihydrofolate reductase (DHFR), have arisen and compromise continued use. In an attempt to extend the lifetime of this important class, we have developed a class of propargyl-linked antifolates (PLAs) that exhibit potent inhibition of the enzyme and bacterial strains. Probing the role of the configuration at the single propargylic stereocenter in these inhibitors required us to develop a new approach to non-racemic 3-aryl-1-butyne building blocks by the pairwise use of asymmetric conjugate addition and aldehyde dehydration protocols. Using this new route, a series of non-racemic PLA inhibitors was prepared and shown to possess potent enzyme inhibition (IC50 values < 50 nM), antibacterial effects (several with MIC values < 1 µg/mL) and to form stable ternary complexes with both wild-type and resistant mutants. Unexpectedly, crystal structures of a pair of individual enantiomers in the wild-type DHFR revealed that the single change in configuration of the stereocenter drove the selection of an alternative NADPH cofactor, with the minor α-anomer appearing with R-27. Remarkably, this cofactor switching becomes much more prevalent when the F98Y mutation is present. The observation of cofactor site plasticity leads to a postulate for the structural basis of TMP resistance in DHFR and also suggests design strategies that can be used to target these resistant enzymes. PMID:26098608

  9. 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.

  10. Relationship between in vitro enhanced nitrogenase activity of an Azospirillum brasilense Sp7 mutant and its growth-promoting activities in situ.

    PubMed

    de Campos, Samanta Bolzan; Roesch, Luiz Fernando Wurdig; Zanettini, Maria Helena Bodanese; Passaglia, Luciane Maria Pereira

    2006-07-01

    In this work, we further analyzed an Azospirillum brasilense Sp7 mutant (Sp7::Tn5-33) showing a pleiotrophic phenotype due to a Tn5 insertion into an open reading frame of 840 bp (orf280). The deduced amino acid sequence of this region has high similarity to a family of universal stress proteins. Because the most interesting property exhibited by the Sp7::Tn5-33 mutant was an enhanced in vitro nitrogen fixation activity, we addressed the question of whether it could benefit the host plant. We found that the increased nitrogenase activity at the free-living state of the mutant bacterium was correlated with an increased production of the nitrogenase reductase protein (NifH), in amounts approximately 1.5 times higher than the wild type. The mutant strain exhibited the same level of auxin production and the same colonization pattern of wheat roots as the wild type. We also observed that Sp7::Tn5-33 increased the total plant dry weight, although the N content did not differ significantly between wheat plants inoculated with mutant or wild-type strains.

  11. Neutrino mass matrices with two equalities between the elements or cofactors

    NASA Astrophysics Data System (ADS)

    Dev, S.; Gautam, Radha Raman; Singh, Lal

    2013-04-01

    We study the implications of the existence of two equalities between the elements or cofactors of the neutrino mass matrix. There are 65 structures of this type for each case. Phenomenological implications for unknown parameters like the effective Majorana mass of the electron neutrino and CP-violating phases are examined for the viable cases. To illustrate how such forms of the neutrino mass matrices may be realized, we also present a simple A4 model for one of the classes in each case.

  12. Neutrino mass textures with one vanishing minor and two equal cofactors

    NASA Astrophysics Data System (ADS)

    Wang, Weijian

    2013-09-01

    In this paper, we carry out a numerical and systematic analysis of the neutrino mass textures, which contain one vanishing minor and equality between two cofactors. Among 60 logically possible textures, only eight of them are excluded for both the normal and inverted hierarchy by the current experimental data at 3 σ level. We also demonstrate that the future long-baseline neutrino oscillation experiments, especially for the measurement of the θ 23 mixing angle, will play an important role in the model selection. The phenomenological implications from neutrinoless double-beta decay and the cosmology observation are also examined. A discussion of the flavor symmetry realization of the textures is also given.

  13. Substituted quinoline quinones as surrogates for the PQQ cofactor: an electrochemical and computational study.

    PubMed

    Dorfner, Walter L; Carroll, Patrick J; Schelter, Eric J

    2015-04-17

    Pyrroloquinoline quinones (PQQ) are important cofactors that shuttle redox equivalents in diverse metalloproteins. Quinoline 7,8-quinones have been synthesized and characterized as surrogates for PQQ to elucidate redox energetics within metalloenzyme active sites. The quinoline 7,8-quinones were accessed using polymer-supported iodoxybenzoic acid and the compounds evaluated using solution electrochemistry. Together with a family of quinones, the products were evaluated computationally and used to generate a predictive correlation between a computed ΔG and the experimental reduction potentials.

  14. The Essential Cofactor TRRAP Recruits the Histone Acetyltransferase hGCN5 to c-Myc

    PubMed Central

    McMahon, Steven B.; Wood, Marcelo A.; Cole, Michael D.

    2000-01-01

    The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation. PMID:10611234

  15. High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases.

    PubMed

    Leidel, Nils; Popović-Bijelić, Ana; Havelius, Kajsa G V; Chernev, Petko; Voevodskaya, Nina; Gräslund, Astrid; Haumann, Michael

    2012-03-01

    Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1Å) and Mn,Fe(III)Fe(II) species (~3.3-3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1. PMID:22222354

  16. Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency.

    PubMed

    Mills, Philippa B; Footitt, Emma J; Ceyhan, Serkan; Waters, Paula J; Jakobs, Cornelis; Clayton, Peter T; Struys, Eduard A

    2012-11-01

    Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

  17. Substrate, Product, and Cofactor: the Extraordinarily Flexible Relationship between the CDE Superfamily and Heme

    PubMed Central

    Celis, Arianna I.; DuBois, Jennifer L.

    2015-01-01

    PFam Clan 0032, also known as the CDE superfamily, is a diverse group of at least 20 protein families sharing a common α, β-barrel domain. Of these, six different groups bind heme inside the barrel’s interior, using it alternately as a cofactor, substrate, or product. Focusing on these six, an integrated picture of structure, sequence, taxonomy, and mechanism is presented here, detailing how a single structural motif might be able to mediate such an array of functions with one of nature’s most important small molecules. PMID:25778630

  18. Cofactor-free detection of phosphatidylserine with cyclic peptides mimicking lactadherin.

    PubMed

    Zheng, Hong; Wang, Fang; Wang, Qin; Gao, Jianmin

    2011-10-01

    Cyclic peptides (cLacs) are designed to mimic the natural phosphatidylserine (PS) binding protein lactadherin. Unlike annexin V or its small molecule mimics, the cLac peptides selectively target PS-presenting membranes with no need for metal cofactors. We further show that a fluorophore-labeled cLac effectively stains early apoptotic cells. The small size and facile conjugation with a variety of imaging tracers make the cLac design promising for imaging cell death in vitro as well as in living organisms.

  19. Binding of dinitrogen to an iron-sulfur-carbon site

    NASA Astrophysics Data System (ADS)

    Čorić, Ilija; Mercado, Brandon Q.; Bill, Eckhard; Vinyard, David J.; Holland, Patrick L.

    2015-10-01

    Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

  20. Binding of dinitrogen to an iron-sulfur-carbon site.

    PubMed

    Čorić, Ilija; Mercado, Brandon Q; Bill, Eckhard; Vinyard, David J; Holland, Patrick L

    2015-10-01

    Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

  1. Conformational control of cofactors in nature - the influence of protein-induced macrocycle distortion on the biological function of tetrapyrroles.

    PubMed

    Senge, Mathias O; MacGowan, Stuart A; O'Brien, Jessica M

    2015-12-14

    Tetrapyrrole-containing proteins are one of the most fundamental classes of enzymes in nature and it remains an open question to give a chemical rationale for the multitude of biological reactions that can be catalyzed by these pigment-protein complexes. There are many fundamental processes where the same (i.e., chemically identical) porphyrin cofactor is involved in chemically quite distinct reactions. For example, heme is the active cofactor for oxygen transport and storage (hemoglobin, myoglobin) and for the incorporation of molecular oxygen in organic substrates (cytochrome P450). It is involved in the terminal oxidation (cytochrome c oxidase) and the metabolism of H2O2 (catalases and peroxidases) and catalyzes various electron transfer reactions in cytochromes. Likewise, in photosynthesis the same chlorophyll cofactor may function as a reaction center pigment (charge separation) or as an accessory pigment (exciton transfer) in light harvesting complexes (e.g., chlorophyll a). Whilst differences in the apoprotein sequences alone cannot explain the often drastic differences in physicochemical properties encountered for the same cofactor in diverse protein complexes, a critical factor for all biological functions must be the close structural interplay between bound cofactors and the respective apoprotein in addition to factors such as hydrogen bonding or electronic effects. Here, we explore how nature can use the same chemical molecule as a cofactor for chemically distinct reactions using the concept of conformational flexibility of tetrapyrroles. The multifaceted roles of tetrapyrroles are discussed in the context of the current knowledge on distorted porphyrins. Contemporary analytical methods now allow a more quantitative look at cofactors in protein complexes and the development of the field is illustrated by case studies on hemeproteins and photosynthetic complexes. Specific tetrapyrrole conformations are now used to prepare bioengineered designer proteins

  2. Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity.

    PubMed

    Stiti, Naim; Podgórska, Karolina; Bartels, Dorothea

    2014-03-01

    The cofactor-binding site of the NAD(+)-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2'- and 3'-hydroxyl groups of the adenosyl ribose ring of NAD(+) and repels the 2'-phosphate moiety of NADP(+). If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD(+) is altered and rendered the enzyme capable of using NADP(+). This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2'-phosphate of NADP(+). Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP(+). The double mutant ALDH3H1Glu149Thr/Ile200Val showed a good catalysis with NADP(+). Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a "closed" cofactor binding site. The cofactor specificity was shifted even further in favor of NADP(+), as the mutant ALDH3H1E149T/V178R/I200V uses NADP(+) with almost 7-fold higher catalytic efficiency compared to NAD(+). Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.

  3. Conformational control of cofactors in nature - the influence of protein-induced macrocycle distortion on the biological function of tetrapyrroles.

    PubMed

    Senge, Mathias O; MacGowan, Stuart A; O'Brien, Jessica M

    2015-12-14

    Tetrapyrrole-containing proteins are one of the most fundamental classes of enzymes in nature and it remains an open question to give a chemical rationale for the multitude of biological reactions that can be catalyzed by these pigment-protein complexes. There are many fundamental processes where the same (i.e., chemically identical) porphyrin cofactor is involved in chemically quite distinct reactions. For example, heme is the active cofactor for oxygen transport and storage (hemoglobin, myoglobin) and for the incorporation of molecular oxygen in organic substrates (cytochrome P450). It is involved in the terminal oxidation (cytochrome c oxidase) and the metabolism of H2O2 (catalases and peroxidases) and catalyzes various electron transfer reactions in cytochromes. Likewise, in photosynthesis the same chlorophyll cofactor may function as a reaction center pigment (charge separation) or as an accessory pigment (exciton transfer) in light harvesting complexes (e.g., chlorophyll a). Whilst differences in the apoprotein sequences alone cannot explain the often drastic differences in physicochemical properties encountered for the same cofactor in diverse protein complexes, a critical factor for all biological functions must be the close structural interplay between bound cofactors and the respective apoprotein in addition to factors such as hydrogen bonding or electronic effects. Here, we explore how nature can use the same chemical molecule as a cofactor for chemically distinct reactions using the concept of conformational flexibility of tetrapyrroles. The multifaceted roles of tetrapyrroles are discussed in the context of the current knowledge on distorted porphyrins. Contemporary analytical methods now allow a more quantitative look at cofactors in protein complexes and the development of the field is illustrated by case studies on hemeproteins and photosynthetic complexes. Specific tetrapyrrole conformations are now used to prepare bioengineered designer proteins

  4. Electronic structural flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

    PubMed

    Shafaat, Hannah S; Griese, Julia J; Pantazis, Dimitrios A; Roos, Katarina; Andersson, Charlotta S; Popović-Bijelić, Ana; Gräslund, Astrid; Siegbahn, Per E M; Neese, Frank; Lubitz, Wolfgang; Högbom, Martin; Cox, Nicholas

    2014-09-24

    The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform. PMID:25153930

  5. Neutrino mass matrices with two vanishing cofactors and Fritzsch texture for charged lepton mass matrix

    NASA Astrophysics Data System (ADS)

    Wang, Weijian; Guo, Shu-Yuan; Wang, Zhi-Gang

    2016-04-01

    In this paper, we study the cofactor 2 zero neutrino mass matrices with the Fritzsch-type structure in charged lepton mass matrix (CLMM). In the numerical analysis, we perform a scan over the parameter space of all the 15 possible patterns to get a large sample of viable scattering points. Among the 15 possible patterns, three of them can accommodate the latest lepton mixing and neutrino mass data. We compare the predictions of the allowed patterns with their counterparts with diagonal CLMM. In this case, the severe cosmology bound on the neutrino mass set a strong constraint on the parameter space, rendering two patterns only marginally allowed. The Fritzsch-type CLMM will have impact on the viable parameter space and give rise to different phenomenological predictions. Each allowed pattern predicts the strong correlations between physical variables, which is essential for model selection and can be probed in future experiments. It is found that under the no-diagonal CLMM, the cofactor zeros structure in neutrino mass matrix is unstable as the running of renormalization group (RG) from seesaw scale to the electroweak scale. A way out of the problem is to propose the flavor symmetry under the models with a TeV seesaw scale. The inverse seesaw model and a loop-induced model are given as two examples.

  6. Effect of mitochondrial cofactors and antioxidants supplementation on cognition in the aged canine.

    PubMed

    Snigdha, Shikha; de Rivera, Christina; Milgram, Norton W; Cotman, Carl W

    2016-01-01

    A growing body of research has focused on modifiable risk factors for prevention and attenuation of cognitive decline in aging. This has led to an unprecedented interest in the relationship between diet and cognitive function. Several preclinical and epidemiologic studies suggest that dietary intervention can be used to improve cognitive function but randomized controlled trials are increasingly failing to replicate these findings. Here, we use a canine model of aging to evaluate the effects of specific components of diet supplementation which contain both antioxidants and a combination of mitochondrial cofactors (lipoic acid [LA] and acetyl-l-carnitine) on a battery of cognitive functions. Our data suggest that supplementation with mitochondrial cofactors, but not LA or antioxidant alone, selectively improve long-term recall in aged canines. Furthermore, we found evidence that LA alone could have cognitive impairing effects. These results contrast to those of a previous longitudinal study in aged canine. Our data demonstrate that one reason for this difference may be the nutritional status of animals at baseline for the 2 studies. Overall, this study suggests that social, cognitive, and physical activity together with optimal dietary intake (rather than diet alone) promotes successful brain aging.

  7. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

    SciTech Connect

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2010-12-03

    The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 {angstrom}, {beta} = 104.9{sup o}; the crystal diffracted to a resolution of 1.9 {angstrom}. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 {angstrom}, and diffracted to 1.75 {angstrom} resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2{sub 1} and R32, respectively.

  8. Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor

    SciTech Connect

    Mirza, I.; Yachnin, B; Wang, S; Grosse, S; Bergeron, H; Imura, A; Iwaki, H; Hasegawa, Y; Lau, P; Berghuis, A

    2009-01-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O{sub 2} as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 {angstrom}. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  9. FAD synthesis and degradation in the nucleus create a local flavin cofactor pool.

    PubMed

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-10-01

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events. PMID:23946482

  10. FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool*

    PubMed Central

    Giancaspero, Teresa Anna; Busco, Giovanni; Panebianco, Concetta; Carmone, Claudia; Miccolis, Angelica; Liuzzi, Grazia Maria; Colella, Matilde; Barile, Maria

    2013-01-01

    FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg−1 protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min−1·mg−1 protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events. PMID:23946482

  11. Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.

    PubMed

    Dubey, Nidhi C; Tripathi, Bijay P; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2016-05-01

    We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support.

  12. Convenient synthesis of deazaflavin cofactor FO and its activity in F(420)-dependent NADP reductase.

    PubMed

    Hossain, Mohammad S; Le, Cuong Q; Joseph, Ebenezer; Nguyen, Toan Q; Johnson-Winters, Kayunta; Foss, Frank W

    2015-05-14

    F420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)(+). For example, F420-dependent NADP(+) oxidoreductase (Fno) is critical to the conversion of CO2 to CH4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F420. Investigating the FO-dependent NADP(+)/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a Km of 4.00 ± 0.39 μM and a kcat of 5.27 ± 0.14 s(-1). The preparation of FO should enable future biochemical studies and novel uses of F420 mimics.

  13. PRIC295, a Nuclear Receptor Coactivator, Identified from PPARα-Interacting Cofactor Complex

    PubMed Central

    Pyper, Sean R.; Viswakarma, Navin; Jia, Yuzhi; Zhu, Yi-Jun; Fondell, Joseph D.; Reddy, Janardan K.

    2010-01-01

    The peroxisome proliferator-activated receptor-α (PPARα) plays a key role in lipid metabolism and energy combustion. Chronic activation of PPARα in rodents leads to the development of hepatocellular carcinomas. The ability of PPARα to induce expression of its target genes depends on Mediator, an evolutionarily conserved complex of cofactors and, in particular, the subunit 1 (Med1) of this complex. Here, we report the identification and characterization of PPARα-interacting cofactor (PRIC)-295 (PRIC295), a novel coactivator protein, and show that it interacts with the Med1 and Med24 subunits of the Mediator complex. PRIC295 contains 10 LXXLL signature motifs that facilitate nuclear receptor binding and interacts with PPARα and five other members of the nuclear receptor superfamily in a ligand-dependent manner. PRIC295 enhances the transactivation function of PPARα, PPARγ, and ERα. These data demonstrate that PRIC295 interacts with nuclear receptors such as PPARα and functions as a transcription coactivator under in vitro conditions and may play an important role in mediating the effects in vivo as a member of the PRIC complex with Med1 and Med24. PMID:20885938

  14. Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2003-11-11

    Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. PMID:14596623

  15. Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2004-10-12

    We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor. PMID:15461458

  16. Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications.

    PubMed

    Dubey, Nidhi C; Tripathi, Bijay P; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2016-05-01

    We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (Km, kcat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (kcat) for PL, while the kcat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support. PMID:27010819

  17. Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions.

    PubMed

    Kocabas, Evren; Liu, Hualan; Hernick, Marcy

    2015-08-01

    Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable K(D)(ZnII) values (~10-20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe(2+) when purified under anaerobic conditions, and Zn(2+) when purified under aerobic conditions. Consequently, Mca is likely a Fe(2+)-dependent enzyme under physiological conditions; with Zn(2+)-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe(2+) form of the enzyme. PMID:26044118

  18. Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG

    PubMed Central

    Tsang, Alice P.; Fujiwara, Yuko; Hom, Dennis B.; Orkin, Stuart H.

    1998-01-01

    GATA transcription factors are required for the differentiation of diverse cell types in several species. Recent evidence suggests that their biologic activities may be modulated through interaction with multitype zinc finger proteins, such as Friend of GATA-1 (FOG) and U-shaped (Ush). In cell culture, FOG cooperates with the hematopoietic transcription factor GATA-1 to promote erythroid and megakaryocytic differentiation. We show here that mice lacking FOG die during mid-embryonic development with severe anemia. FOG−/− erythroid cells display a marked, but partial, blockage of maturation, reminiscent of GATA-1− erythroid precursors. In contrast to GATA-1 deficiency, however, megakaryocytes fail to develop in the absence of FOG. Although the FOG−/− erythroid phenotype supports the proposed role of FOG as a GATA-1 cofactor in vivo, the latter finding points to a pivotal, GATA-1-independent requirement for FOG in megakaryocyte development from the bipotential erythroid/megakaryocytic progenitor. We speculate that FOG and other FOG-like proteins serve as complex cofactors that act through both GATA-dependent and GATA-independent mechanisms. PMID:9553047

  19. A simple method to engineer a protein-derived redox cofactor for catalysis.

    PubMed

    Shin, Sooim; Choi, Moonsung; Williamson, Heather R; Davidson, Victor L

    2014-10-01

    The 6×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co(2+). Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co(2+)-loaded 6×His-tag could substitute for the hemes in the H2O2-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co(2+)-loaded 6×His-tag could mediate long range electron transfer, it was shown that addition of H2O2 to the Co(2+)-loaded 6×His-tagged Cu(1+) amicyanin oxidizes the copper site which is 20Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.

  20. Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions.

    PubMed

    Kocabas, Evren; Liu, Hualan; Hernick, Marcy

    2015-08-01

    Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable K(D)(ZnII) values (~10-20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe(2+) when purified under anaerobic conditions, and Zn(2+) when purified under aerobic conditions. Consequently, Mca is likely a Fe(2+)-dependent enzyme under physiological conditions; with Zn(2+)-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe(2+) form of the enzyme.

  1. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501.

    PubMed

    Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

    2016-07-26

    Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks.

  2. The influence of oxygen on [NiFe]-hydrogenase cofactor biosynthesis and how ligation of carbon monoxide precedes cyanation.

    PubMed

    Stripp, Sven T; Lindenstrauss, Ute; Granich, Claudia; Sawers, R Gary; Soboh, Basem

    2014-01-01

    The class of [NiFe]-hydrogenases is characterized by a bimetallic cofactor comprising low-spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN-) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC-HypD complex acts as a 'construction site' for the Fe-(CN)2CO portion of the cofactor. The order of addition of the CO and CN- ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT-IR spectroscopic analysis of HypC-HypD isolated from a hydrogenase-competent wild-type strain of Escherichia coli. In contrast to previously reported samples, HypC-HypD showed spectral contributions indicative of an electron-rich Fe-CO cofactor, at the same time lacking any Fe-CN- signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O2. Binding of CO protects the site against loss of spectral features associated with O2 damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O2 on in vivo cofactor biosynthesis.

  3. Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification.

    PubMed

    Dougherty, Edward J; Guo, Chunhua; Simons, S Stoney; Chow, Carson C

    2012-01-01

    Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available.

  4. The Influence of Oxygen on [NiFe]–Hydrogenase Cofactor Biosynthesis and How Ligation of Carbon Monoxide Precedes Cyanation

    PubMed Central

    Stripp, Sven T.; Lindenstrauss, Ute; Granich, Claudia; Sawers, R. Gary; Soboh, Basem

    2014-01-01

    The class of [NiFe]–hydrogenases is characterized by a bimetallic cofactor comprising low–spin nickel and iron ions, the latter of which is modified with a single carbon monoxide (CO) and two cyanide (CN−) molecules. Generation of these ligands in vivo requires a complex maturation apparatus in which the HypC–HypD complex acts as a ‘construction site’ for the Fe–(CN)2CO portion of the cofactor. The order of addition of the CO and CN– ligands determines the ultimate structure and catalytic efficiency of the cofactor; however much debate surrounds the succession of events. Here, we present an FT–IR spectroscopic analysis of HypC–HypD isolated from a hydrogenase–competent wild–type strain of Escherichia coli. In contrast to previously reported samples, HypC–HypD showed spectral contributions indicative of an electron–rich Fe–CO cofactor, at the same time lacking any Fe–CN– signatures. This immature iron site binds external CO and undergoes oxidative damage when in contact with O2. Binding of CO protects the site against loss of spectral features associated with O2 damage. Our findings strongly suggest that CO ligation precedes cyanation in vivo. Furthermore, the results provide a rationale for the deleterious effects of O2 on in vivo cofactor biosynthesis. PMID:25211029

  5. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis.

    PubMed

    Chen, Cynthia; Lodish, Harvey F

    2014-06-01

    Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

  6. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

    PubMed Central

    Chen, Cynthia; Lodish, Harvey F.

    2014-01-01

    Key transcriptional regulators of terminal erythropoiesis, such as GATA1 and TAL1, have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation and whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor Tfdp2 were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression, and knockdown of Tfdp2 results in significantly reduced rates of proliferation, as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis. PMID:24607859

  7. Enzymatic synthesis of L-lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle.

    PubMed

    Tong, Xiaodong; El-Zahab, Bilal; Zhao, Xueyan; Liu, Youyan; Wang, Ping

    2011-02-01

    Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L-lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio-based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system.

  8. Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.

    PubMed

    Schmid, Benedikt; Einsle, Oliver; Chiu, Hsiu-Ju; Willing, Andreas; Yoshida, Mika; Howard, James B; Rees, Douglas C

    2002-12-31

    The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP

  9. Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes.

    PubMed

    Tsai, Yi-Chin C; Mueller-Cajar, Oliver; Saschenbrecker, Sandra; Hartl, F Ulrich; Hayer-Hartl, Manajit

    2012-06-01

    The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

  10. Structural basis for double cofactor specificity in a new formate dehydrogenase from the acidobacterium Granulicella mallensis MP5ACTX8.

    PubMed

    Fogal, Stefano; Beneventi, Elisa; Cendron, Laura; Bergantino, Elisabetta

    2015-11-01

    Formate dehydrogenases (FDHs) are considered particularly useful enzymes in biocatalysis when the regeneration of the cofactor NAD(P)H is required, that is, in chiral synthesis with dehydrogenases. Their utilization is however limited to the recycling of NAD(+), since all (apart one) of the FDHs characterized so far are strictly specific for this cofactor, and this is a major drawback for their otherwise wide applicability. Despite the many attempts performed to modify cofactor specificity by protein engineering different NAD(+)-dependent FDHs, in the general practice, glucose or phosphite dehydrogenases are chosen for the recycling of NADP(+). We report on the functional and structural characterization of a new FDH, GraFDH, identified by mining the genome of the extremophile prokaryote Granulicella mallensis MP5ACTX8. The new enzyme displays a valuable stability in the presence of many organic cosolvents as well as double cofactor specificity, with NADP(+) preferred over NAD(+) at acidic pH values, at which it also shows the highest stability. The quite low affinities for both cofactors as well as for the substrate formate indicate, however, that the native enzyme requires optimization to be applied as biocatalytic tool. We also determined the crystal structure of GraFDH both as apoprotein and as holoprotein, either in complex with NAD(+) or NADP(+). Noticeably, the latter represents the first structure of an FDH enzyme in complex with NADP(+). This fine picture of the structural determinants involved in cofactor selectivity will possibly boost protein engineering of the new enzyme or other homolog FDHs in view of their biocatalytic exploitation for NADP(+) recycling.

  11. Structural basis for double cofactor specificity in a new formate dehydrogenase from the acidobacterium Granulicella mallensis MP5ACTX8.

    PubMed

    Fogal, Stefano; Beneventi, Elisa; Cendron, Laura; Bergantino, Elisabetta

    2015-11-01

    Formate dehydrogenases (FDHs) are considered particularly useful enzymes in biocatalysis when the regeneration of the cofactor NAD(P)H is required, that is, in chiral synthesis with dehydrogenases. Their utilization is however limited to the recycling of NAD(+), since all (apart one) of the FDHs characterized so far are strictly specific for this cofactor, and this is a major drawback for their otherwise wide applicability. Despite the many attempts performed to modify cofactor specificity by protein engineering different NAD(+)-dependent FDHs, in the general practice, glucose or phosphite dehydrogenases are chosen for the recycling of NADP(+). We report on the functional and structural characterization of a new FDH, GraFDH, identified by mining the genome of the extremophile prokaryote Granulicella mallensis MP5ACTX8. The new enzyme displays a valuable stability in the presence of many organic cosolvents as well as double cofactor specificity, with NADP(+) preferred over NAD(+) at acidic pH values, at which it also shows the highest stability. The quite low affinities for both cofactors as well as for the substrate formate indicate, however, that the native enzyme requires optimization to be applied as biocatalytic tool. We also determined the crystal structure of GraFDH both as apoprotein and as holoprotein, either in complex with NAD(+) or NADP(+). Noticeably, the latter represents the first structure of an FDH enzyme in complex with NADP(+). This fine picture of the structural determinants involved in cofactor selectivity will possibly boost protein engineering of the new enzyme or other homolog FDHs in view of their biocatalytic exploitation for NADP(+) recycling. PMID:26104866

  12. Exogenous cofactors for the improvement of bioremoval and biotransformation of sulfamethoxazole by Alcaligenes faecalis.

    PubMed

    Zhang, Yi-Bi; Zhou, Jiao; Xu, Qiu-Man; Cheng, Jing-Sheng; Luo, Yu-Lu; Yuan, Ying-Jin

    2016-09-15

    Sulfamethoxazole (SMX), an extensively prescribed or administered antibiotic pharmaceutical product, is usually detected in aquatic environments, because of its incomplete metabolism and elimination. This study investigated the effects of exogenous cofactors on the bioremoval and biotransformation of SMX by Alcaligenes faecalis. High concentration (100mg·L(-1)) of exogenous vitamin C (VC), vitamin B6 (VB6) and oxidized glutathione (GSSG) enhanced SMX bioremoval, while the additions of vitamin B2 (VB2) and vitamin B12 (VB12) did not significantly alter the SMX removal efficiency. Globally, cellular growth of A. faecalis and SMX removal both initially increased and then gradually decreased, indicating that SMX bioremoval is likely dependent on the primary biomass activity of A. faecalis. The decreases in the SMX removal efficiency indicated that some metabolites of SMX might be transformed into parent compound at the last stage of incubation. Two transformation products of SMX, N-hydroxy sulfamethoxazole (HO-SMX) and N4-acetyl sulfamethoxazole (Ac-SMX), were identified by a high-performance liquid chromatograph coupled with mass spectrometer. High concentrations of VC, nicotinamide adenine dinucleotide hydrogen (NADH, 7.1mg·L(-1)), and nicotinamide adenine dinucleotide (NAD(+), 6.6mg·L(-1)), and low concentrations of reduced glutathione (GSH, 0.1 and 10mg·L(-1)) and VB2 (1mg·L(-1)) remarkably increased the formation of HO-SMX, while VB12 showed opposite effects on HO-SMX formation. In addition, low concentrations of GSH and NADH enhanced Ac-SMX formation by the addition of A. faecalis, whereas cofactors (VC, VB2, VB12, NAD(+), and GSSG) had no obvious impact on the formation of Ac-SMX compared with the controls. The levels of Ac-SMX were stable when biomass of A. faecalis gradually decreased, indicating the direct effect of biomass on the formation of Ac-SMX by A. faecalis. In sum, these results help us understand the roles played by exogenous cofactors in

  13. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates.

    PubMed

    Scott, Deborah J; da Costa, Bernardo M T; Espy, Stephanie C; Keasling, Jay D; Cornish, Katrina

    2003-09-01

    Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) cofactor requirements, and are membrane-bound enzymes. PMID:12946411

  14. Exogenous cofactors for the improvement of bioremoval and biotransformation of sulfamethoxazole by Alcaligenes faecalis.

    PubMed

    Zhang, Yi-Bi; Zhou, Jiao; Xu, Qiu-Man; Cheng, Jing-Sheng; Luo, Yu-Lu; Yuan, Ying-Jin

    2016-09-15

    Sulfamethoxazole (SMX), an extensively prescribed or administered antibiotic pharmaceutical product, is usually detected in aquatic environments, because of its incomplete metabolism and elimination. This study investigated the effects of exogenous cofactors on the bioremoval and biotransformation of SMX by Alcaligenes faecalis. High concentration (100mg·L(-1)) of exogenous vitamin C (VC), vitamin B6 (VB6) and oxidized glutathione (GSSG) enhanced SMX bioremoval, while the additions of vitamin B2 (VB2) and vitamin B12 (VB12) did not significantly alter the SMX removal efficiency. Globally, cellular growth of A. faecalis and SMX removal both initially increased and then gradually decreased, indicating that SMX bioremoval is likely dependent on the primary biomass activity of A. faecalis. The decreases in the SMX removal efficiency indicated that some metabolites of SMX might be transformed into parent compound at the last stage of incubation. Two transformation products of SMX, N-hydroxy sulfamethoxazole (HO-SMX) and N4-acetyl sulfamethoxazole (Ac-SMX), were identified by a high-performance liquid chromatograph coupled with mass spectrometer. High concentrations of VC, nicotinamide adenine dinucleotide hydrogen (NADH, 7.1mg·L(-1)), and nicotinamide adenine dinucleotide (NAD(+), 6.6mg·L(-1)), and low concentrations of reduced glutathione (GSH, 0.1 and 10mg·L(-1)) and VB2 (1mg·L(-1)) remarkably increased the formation of HO-SMX, while VB12 showed opposite effects on HO-SMX formation. In addition, low concentrations of GSH and NADH enhanced Ac-SMX formation by the addition of A. faecalis, whereas cofactors (VC, VB2, VB12, NAD(+), and GSSG) had no obvious impact on the formation of Ac-SMX compared with the controls. The levels of Ac-SMX were stable when biomass of A. faecalis gradually decreased, indicating the direct effect of biomass on the formation of Ac-SMX by A. faecalis. In sum, these results help us understand the roles played by exogenous cofactors in

  15. Two "unrelated" families of ATP-dependent enzymes share extensive structural similarities about their cofactor binding sites.

    PubMed Central

    Denessiouk, K. A.; Lehtonen, J. V.; Korpela, T.; Johnson, M. S.

    1998-01-01

    Two proteins, D-alanine:D-alanine ligase and cAMP-dependent protein kinase, share a remarkable degree of structural convergence despite having different three-dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super-secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures. PMID:9605318

  16. Electron transfer between the heme bound oxygen and the tetrahydrobiopterin cofactor of nitric oxide synthase: a DFT study

    NASA Astrophysics Data System (ADS)

    Menyhárd, Dóra K.

    2004-07-01

    Nitric oxide is synthesized from L-Arg by nitric oxide synthases (NOSs). DFT calculations carried out in the present study demonstrate that there is direct coupling between the heme bound oxygen and the tetrahydrobiopterin (H 4B) cofactor in the activated state of NOS. Results indicate that radicalization of H 4B causes the coupled reduction of heme bound oxygen. In our model system H 3B rad radical formation is prompted by proton dissociation from the N5 site of the cofactor; spin density is transferred to the heme bound oxygen, which we found in an orientation preconditioned for H abstraction from the substrate.

  17. Esmond E. Snell--the pathfinder of B vitamins and cofactors.

    PubMed

    Hayashi, Hideyuki; Tanase, Sumio; Yagi, Toshiharu

    2010-04-01

    Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

  18. Nitric oxide synthase in cat brain: cofactors--enzyme-substrate interaction.

    PubMed

    Côté, J F; Roberge, A G

    1996-01-01

    Nitric oxide, derived from L-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin (Vmax: 118 instead of 59.4 pmol of citrulline formed .mg of prot.-1 min-1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the Km value for L-arginine was of 11 microM instead of 41 microM in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions.

  19. Crystal Structures of Phosphite Dehydrogenase Provide Insights into Nicotinamide Cofactor Regeneration

    SciTech Connect

    Zou, Yaozhong; Zhang, Houjin; Brunzelle, Joseph S.; Johannes, Tyler W.; Woodyer, Ryan; Hung, John E.; Nair, Nikhil; van der Donk, Wilfred A.; Zhao, Huimin; Nair, Satish K.

    2012-08-21

    The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD{sup +}-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD{sup +} (1.7 {angstrom} resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 {angstrom} resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

  20. RNA with iron(II) as a cofactor catalyses electron transfer

    NASA Astrophysics Data System (ADS)

    Hsiao, Chiaolong; Chou, I.-Chun; Okafor, C. Denise; Bowman, Jessica C.; O'Neill, Eric B.; Athavale, Shreyas S.; Petrov, Anton S.; Hud, Nicholas V.; Wartell, Roger M.; Harvey, Stephen C.; Williams, Loren Dean

    2013-06-01

    Mg2+ is essential for RNA folding and catalysis. However, for the first 1.5 billion years of life on Earth RNA inhabited an anoxic Earth with abundant and benign Fe2+. We hypothesize that Fe2+ was an RNA cofactor when iron was abundant, and was substantially replaced by Mg2+ during a period known as the ‘great oxidation’, brought on by photosynthesis. Here, we demonstrate that reversing this putative metal substitution in an anoxic environment, by removing Mg2+ and replacing it with Fe2+, expands the catalytic repertoire of RNA. Fe2+ can confer on some RNAs a previously uncharacterized ability to catalyse single-electron transfer. We propose that RNA function, in analogy with protein function, can be understood fully only in the context of association with a range of possible metals. The catalysis of electron transfer, requisite for metabolic activity, may have been attenuated in RNA by photosynthesis and the rise of O2.