Construction and characterization of an OHIO-1 beta-lactamase bearing Met69Ile and Gly238Ser mutations.
Amino acid changes that influence activity and resistance to beta-lactams and beta-lactamase inhibitors were explored by constructing the Gly238Ser and Met69Ile-Gly238Ser mutants of the OHIO-1 beta-lactamase, a class A enzyme of the SHV family. The Km values of cefotaxime and ceftazidime for OHIO-1 and Met69Ile beta-lactamases were > or = 500 microM. The Km of cefotaxime for the Gly238Ser beta-lactamase was 26 microM, and that of ceftazidime was 105 microM. The Km of cefotaxime for the Met69Ile-Gly238Ser beta-lactamase was 292 microM, and that of ceftazidime was 392 microM. For the beta-lactamase inhibitors clavulanate and sulbactam, the apparent Ki values for the Met69Ile-Gly238Ser enzyme were 0.03 and 0.15 microM, respectively. Relative Vmax values indicate that the Met69Ile-Gly238Ser mutant of the OHIO-1 beta-lactamase possesses cephalosporinase activity similar to that of the Gly238Ser mutant but diminished penicillinase activity. In an Escherichia coli DH5alpha strain that possesses a Met69Ile beta-lactamase of the OHIO-1 family, the added Gly238Ser mutation resulted in a phenotype with qualities that confer resistance to expanded-spectrum cephalosporins and, to a lesser extent, beta-lactamase inhibitors.
Bonomo
R A
RA
Research Service, U.S. Department of Veterans Affairs Medical Center, and Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA. rab14@po.cwru.edu
Knox
J R
JR
Rudin
S D
SD
Shlaes
D M
DM
D018550:Cephalosporin Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D012694:Serine / Q000235:genetics / Q000378:metabolism; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007532:Isoleucine; D012694:Serine; D008715:Methionine; D001618:beta-Lactamases; D005998:Glycine