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Joel H Weiner - One of the best experts on this subject based on the ideXlab platform.
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pyranopterin conformation defines the function of molybdenum and tungsten enzymes
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Richard A. Rothery, Benjamin W Stein, Martin L. Kirk, Matthew Solomonson, Joel H WeinerAbstract:We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state. Enzymes belonging to the bacterial Dimethyl Sulfoxide Reductase (DMSOR) family contain a metal-bis-pyranopterin cofactor, the two pyranopterins of which have distinct conformations, with one similar to the predicted tetrahydro form, and the other similar to the predicted dihydro form. Enzymes containing a single pyranopterin belong to either the xanthine dehydrogenase (XDH) or sulfite oxidase (SUOX) families, and these have pyranopterin conformations similar to those predicted for tetrahydro and dihydro forms, respectively. This work provides keen insight into the roles of pyranopterin conformation and oxidation state in catalysis, redox potential modulation of the metal site, and catalytic function.
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correct assembly of iron sulfur cluster fs0 into escherichia coli Dimethyl Sulfoxide Reductase dmsabc is a prerequisite for molybdenum cofactor insertion
Journal of Biological Chemistry, 2011Co-Authors: Huipo Tang, Richard A. Rothery, James E Voss, Joel H WeinerAbstract:Abstract The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli Dimethyl Sulfoxide Reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = ) in its reduced form, resulting in an EPR spectrum with a peak at g ∼ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of Dimethyl Sulfoxide reduction. Comparison with nitrate Reductase A (NarGHI) indicates that a sequence of residues (18CTVNC22) plays a role in both FS0 and Mo-bisPGD coordination. A DmsAΔN21 mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from 18CATVNCBGSRCCP27 to 18CATYCBGVGCCG26 (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsAR61K mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.
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X-ray absorption spectroscopic characterization of the molybdenum site of Escherichia coli Dimethyl Sulfoxide Reductase.
Inorganic chemistry, 2007Co-Authors: Graham N. George, Richard A. Rothery, Christian J. Doonan, Nasim Boroumand, Joel H WeinerAbstract:Structural studies of Dimethyl Sulfoxide (DMSO) Reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO Reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 A, one Mo=O at 1.71 A, and a longer Mo-O at 1.90 A. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO Reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO Reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.
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the escherichia coli ynfefghi operon encodes polypeptides which are paralogues of Dimethyl Sulfoxide Reductase dmsabc
Archives of Biochemistry and Biophysics, 2003Co-Authors: Shannon P Lubitz, Joel H WeinerAbstract:Abstract The ynfEFGHI operon is a paralogue of the Escherichia coli dmsABC operon. ynfE and ynfF are paralogues of dmsA. ynfG and ynfH are paralogues of dmsB and dmsC, respectively. YnfI (dmsD) has no dms paralogue. YnfE/F and YnfG could be detected by immunoblotting with anti-DmsAB antibodies when expressed under the control of a tac or dms promoter. Cells harbouring ynfFGH on a multicopy plasmid supported anaerobic growth with Dimethyl Sulfoxide (DMSO) as respiratory oxidant in a dmsABC deletion, suggesting that YnfFGH forms a heterotimeric enzyme complex similar to DmsABC. Exchange of DmsC by YnfH (DmsAB-YnfH) resulted in membrane localization, anaerobic growth on DMSO, and binding of 2-n-heptyl 4-hydroxyquinoline-N-oxide, indicating that YnfH was a competent anchor. YnfG can also replace DmsB as the electron transfer subunit and assembled [Fe–S] clusters as judged by electron paramagnetic resonance spectroscopy. YnfE and/or YnfF could not form a functional complex with DmsBC and expression of YnfE prevented the accumulation of YnfFGH.
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investigation of escherichia coli Dimethyl Sulfoxide Reductase assembly and processing in strains defective for the sec independent protein translocation system membrane targeting and translocation
Journal of Biological Chemistry, 2001Co-Authors: Damaraju Sambasivarao, Hannah A Dawson, Guijin Zhang, Gillian Shaw, Joel H WeinerAbstract:Dimethyl Sulfoxide Reductase is a heterotrimeric enzyme (DmsABC) localized to the cytoplasmic surface of the inner membrane. Targeting of the DmsA and DmsB catalytic subunits to the membrane requires the membrane targeting and translocation (Mtt) system. The DmsAB dimer is a member of a family of extrinsic, cytoplasmic facing membrane subunits that require Mtt in order to assemble on the membrane. We show that the MttA2, MttB, and presumably MttA1 but not the MttC proteins are required for targeting DmsAB to the membrane. Unlike other Mtt substrates such as trimethylamine N-oxide Reductase, the soluble cytoplasmic DmsAB dimer that accumulates in the mttdeletions is very labile. Deletion of the mttA2or mttB genes also prevents anaerobic growth on fumarate even though fumarate Reductase does not require Mtt for assembly. This was due to the lethality of membrane insertion of DmsC in the absence of the DmsAB subunits. In the absence of DmsC, DmsAB accumulates in the cytoplasm. A 45-amino acid leader on DmsA is removed during assembly. Processing does not require DmsC but does require Mtt. Translocation of DmsAB to the periplasm is not required for processing. The leader may be cleaved by a novel leader peptidase, or the long DmsA leader may traverse the membrane through the Mtt system resulting in cleavage by the periplasmic leader peptidase I followed by release of DmsA into the cytoplasm.
K V Rajagopalan - One of the best experts on this subject based on the ideXlab platform.
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pulsed epr studies of the exchangeable proton at the molybdenum center of Dimethyl Sulfoxide Reductase
Journal of Biological Inorganic Chemistry, 2003Co-Authors: Arnold M Raitsimring, Kimberly Johnson Nelson, Andrei V Astashkin, Changjian Feng, John H Enemark, K V RajagopalanAbstract:Electron spin echo envelope modulation (ESEEM) spectroscopy has been used to determine the hyperfine (hfi) and quadrupole (nqi) interactions of the exchangeable deuteron (proton) at the Mo(V) site of DMSO Reductase. The data obtained have been translated into structure-related parameters. It was found that isotropic hfi constant of the proton is not unique, but is distributed within a range of 26–36 MHz. From this hfi distribution, a 30°-wide distribution of the OH bond orientations due to a rotation around the Mo-O bond was estimated. The angle between the axes of the nqi and anisotropic hfi tensors was found to be anomalously small in comparison with that expected from the Mo-O-D bond geometry. This peculiarity was attributed to the effect of spin density on the hydroxyl oxygen atom. The orientation of the Mo-OH fragment with respect to the g-frame was determined from the experimental orientations of the nqi and hfi tensor axes and a theoretical evaluation of the anisotropic hfi axis direction. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0393-8. On that page (frame on the left side), a link takes you directly to the supplementary material.
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an active site tyrosine influences the ability of the Dimethyl Sulfoxide Reductase family of molybdopterin enzymes to reduce s oxides
Journal of Biological Chemistry, 2001Co-Authors: Kimberly E Johnson, K V RajagopalanAbstract:Abstract Dimethyl Sulfoxide Reductase (DMSOR), trimethylamine-N-oxide Reductase (TMAOR), and biotin Sulfoxide Reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilizeS-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 → Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me3NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me3NO relative to Dimethyl Sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by Dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.
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the 1 3 a crystal structure of rhodobacter sphaeroides Dimethyl Sulfoxide Reductase reveals two distinct molybdenum coordination environments
Journal of the American Chemical Society, 2000Co-Authors: K Temple, K V Rajagopalan, Hermann SchindelinAbstract:During the past four years, a substantial amount of structural information has been accumulated on the molybdoenzyme Dimethyl Sulfoxide (DMSO) Reductase from purple bacteria. This enzyme contains a...
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resonance raman characterization of biotin Sulfoxide Reductase comparing oxomolybdenum enzymes in the me2so Reductase family
Journal of Biological Chemistry, 2000Co-Authors: Shannon D Garton, Carrie A. Temple, K V Rajagopalan, Michael J Barber, Ish K Dhawan, Michael K JohnsonAbstract:Resonance Raman spectroscopy has been used to define active site structures for oxidized Mo(VI) and reduced Mo(IV) forms of recombinant Rhodobacter sphaeroides biotin Sulfoxide Reductase expressed in Escherichia coli. On the basis of 18O/16O labeling studies involving water and the alternative substrate Dimethyl Sulfoxide and the close correspondence to the resonance Raman spectra previously reported for Dimethyl Sulfoxide Reductase (Garton, S. D., Hilton, J., Oku, H., Crouse, B. R., Rajagopalan, K. V., and Johnson, M. K. (1997) J. Am. Chem. Soc. 119, 12906–12916), vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The results indicate that the enzyme cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining coordinated in both redox states. Direct evidence for an oxygen atom transfer mechanism is provided by18O/16O labeling studies, which show that the terminal oxo group at the molybdenum center is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Biotin Sulfoxide Reductase is not reduced by biotin or the nonphysiological products, Dimethyl sulfide and trimethylamine. However, product-induced changes in the Mo=O stretching frequency provide direct evidence for a product-associated mono-oxo-Mo(VI) catalytic intermediate. The results indicate that biotin Sulfoxide Reductase is thermodynamically tuned to catalyze the Reductase reaction, and a detailed catalytic mechanism is proposed.
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structure of the molybdenum site of Dimethyl Sulfoxide Reductase
Journal of the American Chemical Society, 1999Co-Authors: Graham N. George, James Hilton, Carrie Temple, Roger C Prince, K V RajagopalanAbstract:Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonance (EPR) spectroscopies have been used to probe the metal coordination in oxidized and reduced forms of both wild-type and a site-directed mutant of Rhodobacter sphaeroides Dimethyl Sulfoxide (DMSO) Reductase. We confirm our earlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 1113−1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (MoO) at 1.68 A, four thiolate ligands at 2.44 A, and one oxygen at 1.92 A and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo−S at 2.33 A and two different Mo−O ligands at 2.16 and 1.92 A. Mo(V) EPR indicates the presence of one exchangeable oxygen ligand, most likely an Mo−OH, in the signal-giving species, probably originating from the MoO of the oxidized enzyme (Em8.5(IV/V) = +37 mV, Em8.5(V/VI) = +83 mV). The addition of Dimethyl sulfide, in the reverse of the physiological...
Richard A. Rothery - One of the best experts on this subject based on the ideXlab platform.
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pyranopterin conformation defines the function of molybdenum and tungsten enzymes
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Richard A. Rothery, Benjamin W Stein, Martin L. Kirk, Matthew Solomonson, Joel H WeinerAbstract:We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state. Enzymes belonging to the bacterial Dimethyl Sulfoxide Reductase (DMSOR) family contain a metal-bis-pyranopterin cofactor, the two pyranopterins of which have distinct conformations, with one similar to the predicted tetrahydro form, and the other similar to the predicted dihydro form. Enzymes containing a single pyranopterin belong to either the xanthine dehydrogenase (XDH) or sulfite oxidase (SUOX) families, and these have pyranopterin conformations similar to those predicted for tetrahydro and dihydro forms, respectively. This work provides keen insight into the roles of pyranopterin conformation and oxidation state in catalysis, redox potential modulation of the metal site, and catalytic function.
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correct assembly of iron sulfur cluster fs0 into escherichia coli Dimethyl Sulfoxide Reductase dmsabc is a prerequisite for molybdenum cofactor insertion
Journal of Biological Chemistry, 2011Co-Authors: Huipo Tang, Richard A. Rothery, James E Voss, Joel H WeinerAbstract:Abstract The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli Dimethyl Sulfoxide Reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = ) in its reduced form, resulting in an EPR spectrum with a peak at g ∼ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of Dimethyl Sulfoxide reduction. Comparison with nitrate Reductase A (NarGHI) indicates that a sequence of residues (18CTVNC22) plays a role in both FS0 and Mo-bisPGD coordination. A DmsAΔN21 mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from 18CATVNCBGSRCCP27 to 18CATYCBGVGCCG26 (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsAR61K mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.
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X-ray absorption spectroscopic characterization of the molybdenum site of Escherichia coli Dimethyl Sulfoxide Reductase.
Inorganic chemistry, 2007Co-Authors: Graham N. George, Richard A. Rothery, Christian J. Doonan, Nasim Boroumand, Joel H WeinerAbstract:Structural studies of Dimethyl Sulfoxide (DMSO) Reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO Reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 A, one Mo=O at 1.71 A, and a longer Mo-O at 1.90 A. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO Reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO Reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.
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consequences of removal of a molybdenum ligand dmsa ser 176 of escherichia coli Dimethyl Sulfoxide Reductase
Journal of Biological Chemistry, 1996Co-Authors: Catharine A Trieber, Richard A. Rothery, Joel H WeinerAbstract:We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli Dimethyl Sulfoxide (Me2SO) Reductase (DmsABC). Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me2SO and in vitro benzyl viologen:Me2SO oxidoReductase activity. EPR spectroscopy reveals changes in the line shape and the gav of the Mo(V) signals of the S176A and S176C enzymes. The midpoint potentials (Em,7) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are -15 and -175 mV. The Em,7 of the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide. Titration of the S176C mutant produced several overlapping Mo(V) species occurring at Eh > -150 mV, suggesting heterogeneity in the molybdenum environment. A Mo(V) spectrum was not visible in S176H membranes poised between -435 to 350 mV or oxidized with 200 microM ferricyanide. No differences were detected in the EPR spectra of the reduced [4Fe-4S] clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced [4Fe-4S] clusters.
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Engineering a novel iron-sulfur cluster into the catalytic subunit of Escherichia coli Dimethyl-Sulfoxide Reductase.
The Journal of biological chemistry, 1996Co-Authors: Catharine A Trieber, Richard A. Rothery, Joel H WeinerAbstract:Dimethyl-Sulfoxide Reductase (DmsABC) is a complex [Fe-S] molybdoenzyme that contains four [4Fe-4S] clusters visible by electron paramagnetic resonance (EPR) spectroscopy. The enzyme contains four ferredoxin-like Cys groups in the electron transfer subunit, DmsB, and an additional group of Cys residues in the catalytic subunit, DmsA. Mutagenesis of the second Cys, Cys-38, in the DmsA group to either Ser or Ala promotes assembly of a fifth [Fe-S] cluster into the mutant enzyme. The EPR spectra, the temperature dependences, and the microwave power dependences demonstrate that the new clusters are [3Fe-4S] clusters. The [3Fe-4S] clusters in both of the C38S and C38A mutant enzymes are relatively unstable in redox titrations and have midpoint potentials of approximately 178 and 140 mV. Mutagenesis of the DmsA Cys group to resemble a sequence capable of binding an [4Fe-4S] cluster did not change the cluster type but reduced the amount of the cluster present in this mutant enzyme. This report demonstrates that all four EPR detectable [Fe-S] clusters in the wild-type enzyme are ligated by DmsB. Wild-type DmsA does not ligate an [Fe-S] cluster that is visible by EPR spectroscopy.
Graham N. George - One of the best experts on this subject based on the ideXlab platform.
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X-ray absorption spectroscopy of a quantitatively Mo(V) Dimethyl Sulfoxide Reductase species.
Inorganic chemistry, 2013Co-Authors: M. Jake Pushie, Russ Hille, Julien J. H. Cotelesage, Ganna Lyashenko, Graham N. GeorgeAbstract:Molybdenum K-edge X-ray absorption spectroscopy (XAS) has been used to probe the structure of a Mo(V) species that has been suggested to be a catalytic intermediate in the reaction of Dimethyl Sulfoxide (DMSO) Reductase with the alternative substrate trimethylamine N-oxide (Bennet et al. Eur. J. Biochem. 1994, 255, 321-331; Cobb et al. J. Biol. Chem. 2005, 280, 11007-11017; Mtei, et al. J. Am. Chem. Soc. 2011, 133, 9672-9774). The oxidized Mo(VI) state of DMSO Reductase has previously been structurally characterized as being six coordinate, with four sulfurs from pyranopterin dithiolene molybdenum cofactors, a terminal oxygen ligand, and an additional oxygen coordination from a serine residue. We find the most plausible structure for the Mo(V) active site is a five-coordinate species with four sulfur donors from the two pyranopterin dithiolene ligands, with an average Mo-S bond-length of 2.35 A, plus a single oxygen donor at 1.99 A, very likely from an Mo-OH ligand. Our results thus suggest that the oxygen of the serine residue has dissociated from the metal ion, suggesting hitherto unsuspected flexibility of the active site, and calling into question whether this putative intermediate is catalytically relevant. The relevance to previous Mo(V) electron paramagnetic resonance and other spectroscopic studies on DMSO Reductase is discussed. XAS of an extensively studied Mo(V) form of Rhodobacter sphaeroides DMSO Reductase (the high-g split species) shows that previously suggested structures for the active site are likely incorrect.
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Interaction of product analogues with the active site of rhodobacter sphaeroides Dimethyl Sulfoxide Reductase.
Inorganic chemistry, 2007Co-Authors: Graham N. George, Kimberly Johnson Nelson, Hugh H. Harris, Christian J. DoonanAbstract:We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides Dimethyl Sulfoxide (DMSO) Reductase reduced with trimethylarsine and show that this is structurally analogous to the physiologically relevant Dimethyl sulfide reduced DMSO Reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and Dimethyl sulfide complexes suggests, in turn, that the Dimethyl sulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S−O bond, as previously suggested.
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X-ray absorption spectroscopic characterization of the molybdenum site of Escherichia coli Dimethyl Sulfoxide Reductase.
Inorganic chemistry, 2007Co-Authors: Graham N. George, Richard A. Rothery, Christian J. Doonan, Nasim Boroumand, Joel H WeinerAbstract:Structural studies of Dimethyl Sulfoxide (DMSO) Reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO Reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 A, one Mo=O at 1.71 A, and a longer Mo-O at 1.90 A. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO Reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO Reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.
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structure of the molybdenum site of Dimethyl Sulfoxide Reductase
Journal of the American Chemical Society, 1999Co-Authors: Graham N. George, James Hilton, Carrie Temple, Roger C Prince, K V RajagopalanAbstract:Molybdenum K-edge X-ray absorption and Mo(V) electron paramagentic resonance (EPR) spectroscopies have been used to probe the metal coordination in oxidized and reduced forms of both wild-type and a site-directed mutant of Rhodobacter sphaeroides Dimethyl Sulfoxide (DMSO) Reductase. We confirm our earlier findings (George, G. N.; Hilton, J.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 1113−1117) that the molybdenum site of the oxidized Mo(VI) enzyme possesses one terminal oxygen ligand (MoO) at 1.68 A, four thiolate ligands at 2.44 A, and one oxygen at 1.92 A and that the dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo−S at 2.33 A and two different Mo−O ligands at 2.16 and 1.92 A. Mo(V) EPR indicates the presence of one exchangeable oxygen ligand, most likely an Mo−OH, in the signal-giving species, probably originating from the MoO of the oxidized enzyme (Em8.5(IV/V) = +37 mV, Em8.5(V/VI) = +83 mV). The addition of Dimethyl sulfide, in the reverse of the physiological...
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x ray absorption spectroscopy of Dimethyl Sulfoxide Reductase from rhodobacter sphaeroides
Journal of the American Chemical Society, 1996Co-Authors: Graham N. George, James Hilton, K V RajagopalanAbstract:X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase. The molybdenum site of the oxidized protein possesses a novel Mo(VI) mono-oxo site (MoO at 1.68 A) with additional coordination by approximately four thiolate ligands at 2.44 A and probably one oxygen or nitrogen at 1.92 A. The reduced Mo(IV) form of the enzyme is a des-oxomolybdenum with 3−4 thiolates at 2.33 A and two different Mo−O/N ligands at 2.16 A and 1.92 A. Similarly, the stable Mo(V) glycerol-inhibited species is found to be a des-oxomolybdenum with approximately four thiolate ligands at 2.40 A and (probably) two similarly coordinated oxygen or nitrogen ligands at 1.96 A.
Toshio Satoh - One of the best experts on this subject based on the ideXlab platform.
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gene disruption analysis of dppa isolated as a periplasmic molecular chaperone like protein for folding of Dimethyl Sulfoxide Reductase in rhodobacter sphaeroides f sp denitrificans
Fems Microbiology Letters, 2000Co-Authors: Masahiro Matsuzaki, Isamu Yamamoto, Yuso Kiso, Toshio SatohAbstract:The effect of inactivation of DppA, a dipeptide transport protein identified as a periplasmic molecular chaperone-like protein, on the formation of active Dimethyl Sulfoxide Reductase (DMSOR) was examined in Rhodobacter sphaeroides f. sp. denitrificans. All of the dppA-disrupted mutants produced a normal level of native form of DMSOR and grew by DMSO respiration, indicating that the loss of DppA protein alone had no effect on the formation of active DMSOR. The periplasmic fraction of the dppA-disrupted mutant also had the activity to prevent aggregation of acid-unfolded DMSOR. Two proteins, DctP and BztA, were further identified as the proteins with the activity. Their activities, however, were much lower than that of DppA. These results suggest that several substrate binding proteins might be implicated in the folding of unfolded DMSOR in the periplasm.
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detection of a compact folding intermediate of Dimethyl Sulfoxide Reductase secreted from a molybdenum cofactor deficient mutant of rhodobacter sphaeroides f sp denitrificans
Plant and Cell Physiology, 1997Co-Authors: Masahiro Matsuzaki, Toshio SatohAbstract:All of the nine cysteine residues in Dimethyl Sulfoxide Reductase (OMSOR) exist in reduced thiol form. The unfolded form, which was previously detected in DMSOR proteins secreted by spheroplasts prepared from a molybdenum cofactor-deficient mutant, was also detected in spheroplasts from a wild type strain when iodoacetamide was present, suggesting that DMSOR is secreted first in a reduced and unfolded form. In spheroplasts from the mutant, a new folding intermediate migrating between the unfolded and native forms was additionally detected on non-denaturing gel. This intermediate contained no disulfide bonds, but had a folded compact conformation similar to that of the native form.
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stabilization by groel a molecular chaperone and a periplasmic fraction as well as refolding in the presence of dithiothreitol of acid unfolded Dimethyl Sulfoxide Reductase a periplasmic protein of rhodobacter sphaeroides f sp denitrificans
Plant and Cell Physiology, 1996Co-Authors: Masahiro Matsuzaki, Yoko Yamaguchi, Hideo Masui, Toshio SatohAbstract:The mechanisms of folding of a periplasmic protein was studied in vitro using Dimethyl Sulfoxide Reductase (DMSOR), a periplasmic enzyme of Rhodobacter sphaeroides f. sp. denitrificans. When DMSOR was denatured by acidification to pH 2 at 30°C, the molybdenum cofactor was immediately released and unfolded forms of DMSOR appeared within 2 min. When the acid-unfolded DMSOR has been incubated in refolding buffer (pH 8.0) at 20°C for 2 h, it became almost undetectable after electrophoresis on a non-denaturing gel. This result suggests that the acid-unfolded DMSOR might have aggregated after incubation. The aggregation was suppressed by incubation in the presence of commercial GroEL, a molecular chaperone. When reduced dithiothreitol (DTT) was added to the acid-unfolded forms in the presence of GroEL, some of the DMSOR was converted to the native form, which had the same mobility on a non-denaturing gel as the active emzyme. Non-reducing SDS-polyacrylamide gel electrophoresis of the acid-unfolded forms of DMSOR indicated that the unfolded forms were a mixture of heterogeneous ly folded or misfolded forms and that their forms were converted by DTT to the fully reduced form. The periplasmic fraction of the phototroph was also able to suppress the aggregation of the acid-unfolded DMSOR, and a protein(s) with a molecular mass of about 40 kDa in the periplasm was revealed to have stabilizing activity. It appears that there exists a mechanism whereby the unfolded DMSOR that is secreted into the periplasm is maintained in a non-aggregated and reduced form during folding to the native form.
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Cloning and Nucleotide Sequence of the Gene Encoding Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides f. sp. denitrificans
Bioscience biotechnology and biochemistry, 1995Co-Authors: Isamu Yamamoto, Akira Okubo, Makoto Tachibana, Masahiro Matsuzaki, Naoki Wada, Takeshi Ujiiye, Hideyuki Kajiwara, Yoshihiro Watanabe, Hisashi Hirano, Toshio SatohAbstract:The gene encoding Dimethyl Sulfoxide (DMSO) Reductase, which contains a molybdenum cofactor, of the phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans was isolated using an oligonucleotide probe, which was synthesized based on a internal amino acid sequence of the purified enzyme. The DMSO Reductase gene coded for 822 amino acids (2466 base pairs, Mr = 89,206) as a precursor form having a signal peptide of 42 amino acids. The deduced amino acid sequence had high homology with those of some enzymes containing a molybdenum cofactor: trim ethyl amine N-oxide Reductase (48%), biotin Sulfoxide Reductase (44%), and DMSO Reductase (29%) of Escherichia coli.
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Molybdenum requirement for translocation of Dimethyl Sulfoxide Reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.
Journal of bacteriology, 1991Co-Authors: Y Yoshida, Toshio Satoh, M Takai, S TakamiAbstract:Translocation of Dimethyl Sulfoxide (DMSO) Reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO Reductase accumulated during induction of the Reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO Reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.