Molecular cloning and expression of biotin sulfoxide reductase from Rhodobacter sphaeroides forma sp. denitrificans.
Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R. sphaeroides BSO reductase which has been sequenced. The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop. The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin. When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.
Pollock
V V
VV
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612, USA.
Barber
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D010088:Oxidoreductases; C019792:biotin sulfoxide reductase