Mutations affecting the catalytic activity of Bacillus cereus 5/B/6 beta-lactamase II.
Random in vitro mutagenesis of a cloned Bacillus cereus 5/B/6 beta-lactamase II gene was used to select defective genes unable to confer ampicillin or cephalosporin C resistance to Escherichia coli. DNA sequencing of mutant genes identified histidine at position 28 as important to beta-lactamase II function. In addition, the isolation of six identical frameshift mutants established that the carboxyl-terminal end of beta-lactamase II is critical for enzyme function. Random mutagenesis also revealed that His88 (implicated previously as one of 4 residues acting as a zinc ligand) is crucial to enzymatic activity and that a glycine to glutamic acid substitution at position 148 produced a defective beta-lactamase. Oligonucleotide mutagenesis directed at Glu37 and Glu212 suggests that these residues are inconsequential to enzyme function but that histidine at position 28 may be involved in substrate binding or recognition.
Lim
H M
HM
School of Life and Health Sciences, University of Delaware, Newark 19716.
Pène
J J
JJ
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D002511:Cephalosporins; D004247:DNA / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics*
D002511:Cephalosporins; D004247:DNA; D001618:beta-Lactamases