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 cell biotin sulfoxide reductase and in Rhodobacter sphaereides 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. cell 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. sphaereides 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. cell 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. (C) 1995 Academic Press, Inc.