Biotin Sulfoxide Reductase

Abstract

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 mol of biotin sulfoxide reduced per min/nmol of enzyme and K m values of 29 and 15 M for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.Biotin sulfoxide (BSO) 1 reductase, which catalyzes the reduction of d-biotin d-sulfoxide to d-biotin according to the following scheme,has been postulated to function in a variety of roles in bacterial metabolism (Pierson and Campbell, 1990) including scavenging biotin sulfoxide from the environment and thus allowing bacteria to utilize this oxidized form of biotin for biosynthetic reactions: reducing bound intracellular biotin, such as bound to biotin-containing carboxylases or protein degradation products, that have become oxidized in aerobic environments; or as a potential protector of the cell from oxidative damage similar to the proposed roles of methionine sulfoxide reductase (Ejiri et al., 1980;Brot et al., 1981;Rahman et al., 1992) and superoxide dismutase (Fridovich, 1989).BSO reductase has been partially purified from Escherichia coli and demonstrated to be a soluble protein that requires an unidentified form of the Mo cofactor (Rajagopalan and Johnson, 1992) and several accessory proteins for activity (del CampilloCampbell and Campbell, 1982). These accessory proteins include a small, heat stable, thioredoxin-like protein moiety, referred to as protein-(SH) 2 and which functions as a source of reducing equivalents and an unidentified flavoprotein (del Campillo-Campbell et al., 1979). The extensive characterization of BSO reductase has been limited by the low natural abundance of the protein, its constitutive expression, and the requirement for auxiliary proteins for activity coupled with the difficulty of its detection in the absence of specific antibodies. While the E. coli BSO reductase bisC structural gene has been cloned and sequenced, the enzyme has not been produced using hete...

Resonance Raman Characterization of Biotin Sulfoxide Reductase

“…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.…”

“…Consequently resonance Raman experiments with 18 O-labeled substrate were carried out with an alternative substrate, Me 2 SO (14).…”

“…Sequence comparisons of BSO reductase and Me 2 SO reductase indicate a high degree of homology (50% identity). In particular, the residues involved in interaction with the two molybdopterin guanine dinucleotide units and the serine that ligates Mo are all strictly conserved (13,14). In this work, we report on resonance Raman studies of R. sphaeroides BSO reductase in oxidized and reduced forms in the presence and absence of substrates and products.…”

“…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 sulfoxide (14).…”

“…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 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.…”