Biotin sulfoxide reductase. Heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme.
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.
Pollock
V V
VV
Department of Biochemistry and Molecular Biology, College of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.
Barber
M J
MJ
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011622:Pterins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012242:Rhodobacter sphaeroides; D013056:Spectrophotometry, Ultraviolet
D006150:Guanine Nucleotides; D011622:Pterins; D011993:Recombinant Fusion Proteins; C063761:molybdopterin guanine dinucleotide; D009249:NADP; D010088:Oxidoreductases; C019792:biotin sulfoxide reductase