Mutations altering substrate specificity in OHIO-1, and SHV-1 family beta-lactamase.
The OHIO-1 beta-lactamase does not normally hydrolyse oxyimino-beta-lactam substrates like cefotaxime, ceftriaxone, ceftazidime or aztreonam. We were able to select spontaneous mutants of an OHIO-1-bearing strain of Escherichia coli using the antibiotic substrates listed above by enrichment methods of frequencies of 10(-8)-10(-10) for all antibiotics except ceftazidime (frequency less than 10(-10)). Most mutants with increased resistance to the other beta-lactams were also more resistant to ceftazidime. Mutations identified by DNA sequencing included a Gly238----Ser238 substitution identical with the SHV-2 mutation previously described, cysteine and valine substitutions at the identical site, and a Gly242----Cys242 substitution. The Cys238 and Cys242 mutant enzymes had less affinity for aztreonam than had the other mutant enzymes. Hydrolysis of cefotaxime, but not cephaloridine, by the cysteine-substituted enzymes was inhibited by p-chloromercuribenzoate. The mutant enzymes had, in general, greater affinity for the mechanism-based inhibitors sulbactam, clavulanic acid and tazobactam. These results suggest two non-mutually exclusive hypotheses for the structural role of substitutions in this area of the enzyme. Either potential hydrogen-bond donors, such as serine and cysteine, interact directly with the beta-lactam molecules, or the steric bulk of these substitutions distorts the beta-pleated sheet such that the beta-lactam is held in a position favourable for stable binding and catalysis. Finally, our data raise questions about a strategy relying on oligonucleotide-probe technology to detect such mutations, because of the variety of substitutions that give rise to similar phenotypes.
Shlaes
D M
DM
Medical Service, Veterans Affairs Medical Center, Cleveland, OH.
Currie-McCumber
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D009820:Ohio; D010957:Plasmids; D013379:Substrate Specificity / Q000235:genetics; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D002729:Chloromercuribenzoates; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases