Inactivation of blasticidin S by Bacillus cereus. VI. Structure and comparison of the bsr gene from a blasticidin S-resistant Bacillus cereus.
Two types of recombinant plasmids containing 600 bp Nde I fragments that coded the bsr gene in opposite directions were obtained. Nucleotide sequencing shows that the bsr encodes a 140 amino acid protein with a putative molecular weight of 15560, the same as that of purified blasticidin S (BS)-deaminase (BSR), on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15500). Upstream of the open reading frame, a Shine-Dalgarno (SD) sequence, frequent inverted repeats, and the sigmaA and sigmaB promoter sequences are observed. The transcriptional start point was determined to be the A located 7 bases downstream from the putative sigmaA promoter (91TTGATC and 113TAAAAT) by the primer extension method and site directed mutagenesis at the -10 or -35 promoter region. A comparison of the amino acid sequence of BSR with that of BS-deaminase from Aspergillus terreus (BSD) showed 27.2% homology. Low degrees of homology were also observed with cytidine deaminase and deoxy cytidine monophosphate (CMP) deaminase. Four conserved amino acid motifs were observed, VGAx6G, C(orH)AEx6A, SPCGxCR, and Gx8ELIP (x(n) indicates a nonspecific residue and its position). It is possible that the three Cys residues and the Glu in the conserved motifs comprise the active center. Site-directed mutagenesis of the Cys residues supports this possibility.
Nawa (née Kobayashi)
K
K
Kyoritsu College of Pharmacy, Tokyo, Japan.
Tanaka
T
T
Kamakura
T
T
Yamaguchi
I
I
Endo
T
T
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics* / Q000494:pharmacology*; D001409:Bacillus cereus / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001711:Biotransformation; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000493:pharmacokinetics / Q000494:pharmacology; D009709:Nucleotide Deaminases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009705:Nucleosides; C004500:blasticidin S; D000619:Aminohydrolases; D009709:Nucleotide Deaminases; C019795:blasticidin S deaminase