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 micromol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 microM 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.
Highlights
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
Biotin sulfoxide (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
We have provided the first demonstration that the enzyme can directly utilize reduced pyridine nucleotides as a source of reducing equivalents and obtained initial rate kinetic constants that indicate that BSO reductase can utilize a variety of alternate oxidizing substrates in addition to d-biotin d-sulfoxide
Summary
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. 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) and which functions as a source of reducing equivalents and an unidentified flavoprotein (del Campillo-Campbell et al, 1979). Previous attempts to develop E. coli-based heterologous expression systems for this group of MGD-containing enzymes has been limited to R. sphaeroides Me2SO reductase, where the enzyme was produced in an insoluble and inactive form (Hilton and Rajagopalan, 1996a)
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