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Alasdair M Cook - One of the best experts on this subject based on the ideXlab platform.
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dissimilatory Sulfite Reductase desulfoviridin of the taurine degrading non sulfate reducing bacterium bilophila wadsworthia rzatau contains a fused dsrb dsrd subunit
Journal of Bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory Sulfite Reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing Sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The Sulfite Reductase was isolated as an a2b2gn (n > 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the a and b subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (a) and 54 kDa (b) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6. Bilophila wadsworthia is a strictly anaerobic, gram-negative bacterium (2) which belongs to the family Desulfovibrionaceae in the delta subdivision of the Proteobacteria, but does not
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Dissimilatory Sulfite Reductase (Desulfoviridin) of the Taurine-Degrading, Non-Sulfate-Reducing Bacterium Bilophila wadsworthia RZATAU Contains a Fused DsrB-DsrD Subunit
Journal of bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory Sulfite Reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing Sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The Sulfite Reductase was isolated as an alpha2beta)gamma(n) (n > or = 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the alpha and beta subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (alpha) and 54 kDa (beta) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6.
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a Fused DsrB-DsrD Subunit
2000Co-Authors: Heike Laue, Michael Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory Sulfite Reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing Sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The Sulfite Reductase was isolated as an � 2 � 2 � n (n> 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the � and � subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (�) and 54 kDa (�) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6. Bilophila wadsworthia is a strictly anaerobic, gram-negative bacterium (2) which belongs to the family Desulfovibrionaceae in the delta subdivision of the Proteobacteria, but does no
M. Fontecave - One of the best experts on this subject based on the ideXlab platform.
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four crystal structures of the 60 kda flavoprotein monomer of the Sulfite Reductase indicate a disordered flavodoxin like module
Journal of Molecular Biology, 2000Co-Authors: Arnaud Gruez, M. Fontecave, J Covès, Mahel Zeghouf, David Pignol, Jeanluc Ferrer, Juan C FontecillacampsAbstract:Escherichia coli NADPH-Sulfite Reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of Sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for Sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ Reductase family. By analogy with the structure of the cytochrome P450 Reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 Reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
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the flavoprotein component of the escherichia coli Sulfite Reductase expression purification and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors
Biochemistry, 1998Co-Authors: Mahel Zeghouf, M. Fontecave, David Macherel, J CovèsAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the Sulfite Reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH• is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquin...
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flavin mononucleotide binding domain of the flavoprotein component of the Sulfite Reductase from escherichia coli
Biochemistry, 1997Co-Authors: J Covès, Mahel Zeghouf, David Macherel, Bruno Guigliarelli, Marcel Asso, M. FontecaveAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E‘1 (FMNH•/FMN) = −130 ± 10 mV and E‘2 (FMNH2/FMNH•) = −335 ± 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 2...
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flavin mononucleotide binding domain of the flavoprotein component of the Sulfite Reductase from escherichia coli
Biochemistry, 1997Co-Authors: J Covès, Mahel Zeghouf, David Macherel, Bruno Guigliarelli, Marcel Asso, M. FontecaveAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 Reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.
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nadph Sulfite Reductase flavoprotein from escherichia coli contribution to the flavin content and subunit interaction
FEBS Letters, 1995Co-Authors: M Eschenbrenner, J Covès, M. FontecaveAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase of E. coli is an octamer of the 66 kDa α subunit. It was shown to be cleaved in two peptide fragments. The 23 kDa fragment has been purified as a polymer of 8–10 subunits. It corresponds to the N-terminal part of the native protein and was shown to contain essentially FMN as cofactor. The 43 kDa fragment is monomeric. It contains exclusively FAD and remains able to catalyze efficiently NADPH-dependent reductions. One can conclude that each α-chain of SiR-FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN-binding domains cooperate for a head-to-head subunit interaction.
Michael W Friedrich - One of the best experts on this subject based on the ideXlab platform.
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lateral gene transfer of dissimilatory bi Sulfite Reductase revisited
Journal of Bacteriology, 2005Co-Authors: Alexander Loy, Vladimir V Zverlov, Michael Klein, Sebastian Lucker, Michael W Friedrich, Josef Kellermann, David A Stahl, Michael WagnerAbstract:In contrast to previous findings, we demonstrate that the dissimilatory (bi)Sulfite Reductase genes (dsrAB) of Desulfobacula toluolica were vertically inherited. Furthermore, Desulfobacterium anilini and strain mXyS1 were identified, by dsrAB sequencing of 17 reference strains, as members of the donor lineage for those gram-positive Desulfotomaculum species which laterally acquired dsrAB.
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multiple lateral transfers of dissimilatory Sulfite Reductase genes between major lineages of sulfate reducing prokaryotes
Journal of Bacteriology, 2001Co-Authors: Michael Klein, Michael W Friedrich, David A Stahl, Andrew J Roger, Philip Hugenholtz, Susan Fishbain, Heike Abicht, Linda L Blackall, Michael WagnerAbstract:A large fragment of the dissimilatory Sulfite Reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the Sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with δ-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the δ-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the δ-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.
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dissimilatory Sulfite Reductase desulfoviridin of the taurine degrading non sulfate reducing bacterium bilophila wadsworthia rzatau contains a fused dsrb dsrd subunit
Journal of Bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory Sulfite Reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing Sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The Sulfite Reductase was isolated as an a2b2gn (n > 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the a and b subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (a) and 54 kDa (b) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6. Bilophila wadsworthia is a strictly anaerobic, gram-negative bacterium (2) which belongs to the family Desulfovibrionaceae in the delta subdivision of the Proteobacteria, but does not
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Dissimilatory Sulfite Reductase (Desulfoviridin) of the Taurine-Degrading, Non-Sulfate-Reducing Bacterium Bilophila wadsworthia RZATAU Contains a Fused DsrB-DsrD Subunit
Journal of bacteriology, 2001Co-Authors: Heike Laue, Michael W Friedrich, Jurgen Ruff, Alasdair M CookAbstract:A dissimilatory Sulfite Reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing Sulfite, which is formed during the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The Sulfite Reductase was isolated as an alpha2beta)gamma(n) (n > or = 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the alpha and beta subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrA and dsrB encode polypeptides of 49 (alpha) and 54 kDa (beta) which show significant similarities to the homologous subunits of other DSRs. The dsrB gene product of B. wadsworthia is apparently a fusion protein of dsrB and dsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed that B. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6.
J Covès - One of the best experts on this subject based on the ideXlab platform.
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solution structure of the Sulfite Reductase flavodoxin like domain from escherichia coli
Biochemistry, 2005Co-Authors: Nathalie Sibille, J Covès, Martin Blackledge, Bernhard Brutscher, Beate BerschAbstract:The flavoprotein moiety of Escherichia coli Sulfite Reductase (SiR-FP) is homologous to electron transfer proteins such as cytochrome-P450 Reductase (CPR) or nitric oxide synthase (NOS). We report on the three-dimensional structure of SiR-FP18, the flavodoxin-like domain of SiR-FP, which has been determined by NMR. In the holoenzyme, this domain plays an important role by shuttling electrons from the FAD to the hemoprotein (the beta-subunit). The structure presented here was determined using distance and torsion angle information in combination with residual dipolar couplings determined in two different alignment media. Several protein-FMN NOEs allowed us to place the prosthetic group in its binding pocket. The structure is well-resolved, and (15)N relaxation data indicate that SiR-FP18 is a compact domain. The binding interface with cytochrome c, a nonphysiological electron acceptor, has been determined using chemical shift mapping. Comparison of the SiR-FP18 structure with the corresponding domains from CPR and NOS shows that the fold of the protein core is highly conserved, but the analysis of the electrostatic surfaces reveals significant differences between the three domains. These observations are placed in the physiological context so they can contribute to the understanding of the electron transfer mechanism in the SiR holoenzyme.
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four crystal structures of the 60 kda flavoprotein monomer of the Sulfite Reductase indicate a disordered flavodoxin like module
Journal of Molecular Biology, 2000Co-Authors: Arnaud Gruez, M. Fontecave, J Covès, Mahel Zeghouf, David Pignol, Jeanluc Ferrer, Juan C FontecillacampsAbstract:Escherichia coli NADPH-Sulfite Reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of Sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for Sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ Reductase family. By analogy with the structure of the cytochrome P450 Reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 Reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
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the flavoprotein component of the escherichia coli Sulfite Reductase expression purification and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors
Biochemistry, 1998Co-Authors: Mahel Zeghouf, M. Fontecave, David Macherel, J CovèsAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the Sulfite Reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH• is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquin...
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flavin mononucleotide binding domain of the flavoprotein component of the Sulfite Reductase from escherichia coli
Biochemistry, 1997Co-Authors: J Covès, Mahel Zeghouf, David Macherel, Bruno Guigliarelli, Marcel Asso, M. FontecaveAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E‘1 (FMNH•/FMN) = −130 ± 10 mV and E‘2 (FMNH2/FMNH•) = −335 ± 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 2...
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flavin mononucleotide binding domain of the flavoprotein component of the Sulfite Reductase from escherichia coli
Biochemistry, 1997Co-Authors: J Covès, Mahel Zeghouf, David Macherel, Bruno Guigliarelli, Marcel Asso, M. FontecaveAbstract:The flavoprotein component (SiR-FP) of the Sulfite Reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 Reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.
Michael Wagner - One of the best experts on this subject based on the ideXlab platform.
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reverse dissimilatory Sulfite Reductase as phylogenetic marker for a subgroup of sulfur oxidizing prokaryotes
Environmental Microbiology, 2009Co-Authors: Alexander Loy, Christiane Dahl, Stephan Duller, Christian Baranyi, Marc Musmann, Jorg A Ott, Itai Sharon, Oded Beja, Denis Le Paslier, Michael WagnerAbstract:Summary Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of dsrAB that encode the reversely operating sirohaem dissimilatory Sulfite Reductase (rDSR) and are present in many but not all published genomes of SOP. The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.
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lateral gene transfer of dissimilatory bi Sulfite Reductase revisited
Journal of Bacteriology, 2005Co-Authors: Alexander Loy, Vladimir V Zverlov, Michael Klein, Sebastian Lucker, Michael W Friedrich, Josef Kellermann, David A Stahl, Michael WagnerAbstract:In contrast to previous findings, we demonstrate that the dissimilatory (bi)Sulfite Reductase genes (dsrAB) of Desulfobacula toluolica were vertically inherited. Furthermore, Desulfobacterium anilini and strain mXyS1 were identified, by dsrAB sequencing of 17 reference strains, as members of the donor lineage for those gram-positive Desulfotomaculum species which laterally acquired dsrAB.
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multiple lateral transfers of dissimilatory Sulfite Reductase genes between major lineages of sulfate reducing prokaryotes
Journal of Bacteriology, 2001Co-Authors: Michael Klein, Michael W Friedrich, David A Stahl, Andrew J Roger, Philip Hugenholtz, Susan Fishbain, Heike Abicht, Linda L Blackall, Michael WagnerAbstract:A large fragment of the dissimilatory Sulfite Reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the Sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with δ-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the δ-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the δ-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.
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diversity of sulfate reducing bacteria in oxic and anoxic regions of a microbial mat characterized by comparative analysis of dissimilatory Sulfite Reductase genes
Applied and Environmental Microbiology, 1999Co-Authors: Dror Minz, Michael Wagner, Jodi Flax, Stefan J Green, Gerard Muyzer, Yehuda Cohen, Bruce E Rittmann, David A StahlAbstract:Sequence analysis of genes encoding dissimilatory Sulfite Reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the δ-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.
