Structure of the Molybdenum Site of <i>Rhodobacter sphaeroides</i> Biotin Sulfoxide Reductase

Temple, Carrie A.; George, Graham N.; Hilton, J.; George, Martin J.; Prince, Roger C.; Barber, Michael J.; Rajagopalan, K.V.

doi:10.1021/bi9921541

Cited by 35 publications

(40 citation statements)

References 26 publications

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“…The UV-visible absorption spectra of as prepared and dithionite-reduced recombinant R. sphaeroides BSO reductase are very similar to those of the corresponding samples of wild-type R. sphaeroides Me 2 SO reductase (15). As prepared, BSO reductase has maxima at 350 (⑀ ϭ 5.3 mM…”

Section: Resultsmentioning

confidence: 55%

“…BSO Reductase Samples-Heterologous expression of R. sphaeroides BSO reductase in E. coli has been reported in the literature by Pollock and Barber (14) Resonance Raman samples were prepared using a modified procedure that results in a much greater yield of recombinant enzyme (15). Samples were prepared in 50 mM tricine buffer, pH 8.0, and were concentrated to ϳ4 mM for resonance Raman studies by Centricon ultrafiltration.…”

Section: Methodsmentioning

confidence: 99%

“…Using either reduced methyl viologen or NADPH as electron donor, the enzyme was found to exhibit broad substrate specificity and was able to catalyze reduction of nicotinamide-N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide with varying efficiencies, in addition to the reduction of biotin sulfoxide (14). Recent improvements in the expression system have produced higher yields of recombinant BSO reductase (15), making the enzyme more readily available for extensive spectroscopic characterization.…”

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confidence: 99%

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Resonance Raman Characterization of Biotin Sulfoxide Reductase

Garton

Temple

Dhawan

et al. 2000

Journal of Biological Chemistry

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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 18 With the exception of nitrogenase, molybdenum enzymes catalyze formal oxygen atom transfer between water and substrate and contain an active site in which the molybdenum is coordinated by the dithiolene side chain of one or two molybdopterins (Fig. 1a) (1-3). Although the recent proliferation of x-ray crystal structures for mononuclear molybdenum enzymes has revealed a common structure for the molybdopterin cofactor, they have also revealed considerable diversity in the molybdenum coordination environment (2-11). However, on the basis of the available crystallographic, spectroscopic, primary sequence, and cyanide inhibition data, these enzymes can be divided into three large families (Fig. 1b) (1). Hydroxylases, such as Desulfovibrio gigas aldehyde oxidoreductase, xanthine oxidase, and xanthine dehydrogenase, represent the xanthine oxidase family, characterized by a Mo(VI) active site with a single molybdopterin, as well as terminal oxo and sulfido groups. The oxotransferase class of molybdoenzymes catalyzes direct oxygen atom transfer between substrate and water and can be divided into two families. The sulfite oxidase family, as represented by sulfite oxidase and assimilatory nitrate reductase, contains Mo(VI) ligated by two oxo groups in addition to a single molybdopterin and a cysteine residue. The dimethyl sulfoxide (Me 2 SO) reductase family comprises mono-oxoMo(VI) sites ligated by two molybdopterins and a protein side chain ligand such as serine, cysteine, or selenocysteine. In addition to Me 2 SO reductase, this family also includes formate dehydrogenase, dissimilatory nitrate reductase, and biotin sulfoxide (BSO) 1 reductase. BSO reductase is found in bacterial systems, such as Rhodobacter sphaeroides and Escherichia coli, and catalyzes the reduction of d-biotin d-sulfoxide to d-biotin (Fig. 2). Although the precise role for BSO reductase in bacterial metabolism has yet to be defined, potential physiological functions include scavenging biotin sulfoxide from the environment, reducing bound intracellular biotin that has become oxidized in an aerobic environment, and protecting the cell from oxidative damage (12). Extensive characterization of this enzyme has been limited due to the low natural abundance of the protein and its constitutive expression (13). However, R. sphaeroides BSO reductase has recently been heterologously expressed in E. coli and characterized as containing the molybdopterin guanine dinucleotide form of the molybdopterin cofactor (14). Using either reduced methyl viologen or NADPH as electron donor, the enzyme was found to exhibit broad substrate specificity and was able to catalyze reduction of nicotinamide-N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide with varying efficiencies, in addition to the reduction of biotin ...

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Section: Resultsmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Resonance Raman Characterization of Biotin Sulfoxide Reductase

Garton

Temple

Dhawan

et al. 2000

Journal of Biological Chemistry

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“…Creation of Expression Constructs-Whereas E. coli TMAOR has previously been purified from source (30,31) and a similar enzyme has been purified from S. massilia (32), the yield in both cases was substantially less than that obtained by the heterologous expression of R. sphaeroides DMSOR and BSOR in E. coli (3,28). To facilitate purification of the large quantities of enzyme required for comprehensive studies on the role of the Tyr residue in DMSOR and TMAOR, E. coli TMAOR was cloned, homogeneously expressed, and purified.…”

Section: Resultsmentioning

confidence: 99%

“…Although R. sphaeroides DMSOR and BSOR were previously cloned and heterologously expressed in E. coli (3,23,26), TMAOR has not been cloned previously. In the studies reported here, E. coli TMAOR has been cloned and the recombinant protein purified, setting the stage for a comprehensive study of the role of Tyr-114 in this family of enzymes.…”

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An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

Johnson

Rajagopalan

2001

Journal of Biological Chemistry

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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 utilize S-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 3 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 wildtype enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me 3 NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me 3 NO 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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Mono(maleonitriledithiolene)molybdenum(IV) and Bis(μ‐sulfido)‐Bridged Dimolybdenum(V) Complexes with MoS Moiety

Bose

Moula

Sarkar

2012

Chemistry & Biodiversity

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Mono(maleonitriledithiolene)sulfidomolybdenum(IV) complex, [MoS(S(4))(mnt)](2-) (2; mnt=maleonitriledithiolene) was synthesized by the substitution reaction of a tetrasulfido ligand of the known [MoS(S(4))(2)](2-) (1) upon reaction with one or even excess equivalent of Na(2)(mnt) in aqueous MeCN solution in air. Surprisingly, 2 undergoes dimerization on treatment with alkyl halide such as MeI and PhCH(2)Br to form bis(μ-sulfido)dimolybdenum(V) species, [{MoS(mnt)}(2)(μ-S)(2)](2-) (3). These complexes have been characterized by IR, UV/VIS spectroscopy, cyclic voltammetry, elemental analysis, and by X-ray crystal-structure analysis. Differences in the relative stability and electrochemical behavior of 1, 2, and 3 have been correlated with theoretical calculations at DFT level.

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Structure of the Molybdenum Site of Rhodobacter sphaeroides Biotin Sulfoxide Reductase

Cited by 35 publications

References 26 publications

Resonance Raman Characterization of Biotin Sulfoxide Reductase

Resonance Raman Characterization of Biotin Sulfoxide Reductase

An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides

Mono(maleonitriledithiolene)molybdenum(IV) and Bis(μ‐sulfido)‐Bridged Dimolybdenum(V) Complexes with MoS Moiety

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