MOLECULAR-CLONING AND EXPRESSION OF BIOTIN SULFOXIDE REDUCTASE FROM RHODOBACTER-SPHAEROIDES FORMA SP DENITRIFICANS

Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulf oxide (BSO) to d-biotin, Oligonucleotides directed against common sequ ences in Escherichia cell biotin sulfoxide reductase and in Rhodobacte r sphaereides f.sp, denitrificans dimethyl sulfoxide reductase have be en utilized to amplify by PCR a 651-bp fragment from R. sphaeroides to tal genomic DNA that showed a high degree of sequence similarity with both E. cell biotin sulfoxide reductase and R. sphaeroides dimethyl su lfoxide reductase. Screening of a genomic cosmid library, prepared fro m R. sphaeroides genomic DNA, with this probe resulted in the isolatio n of a 7-kb EcoRI-EcoRI fragment that contained the complete coding re gion for R. sphaereides BSO reductase which has been sequenced, The se quence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subun it molecular weight of 80,234 Da. The translated protein sequence cont ained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor bindin g motifs) and a ATP/GTP-binding P-loop, The R. sphaeroides BSO reducta se sequence showed 51% sequence similarity with the corresponding E. c oli enzyme. In addition, there were only two conserved cysteines betwe en the two BSO reductase sequences. The R. sphaeroides gene was demons trated, by complementation, to rescue a mutant E. cell strain that was deficient in BSO reductase when grown on BSO as the sole source of bi otin, When expressed from the FLAGShift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approxi mately 80 kDa, which cross-reacted with the anti-FLAG: monoclonal anti body and exhibited BSO reductase activity by the disk microbiological assay. (C) 1995 Academic Press, Inc.