OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.
Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.
Lin
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Research Service, Department of Veterans Affairs Medical Center, Department of Medicine, Case Western Reserve University, 10701 East Blvd., Cleveland, OH 44106, USA.
Thomas
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Mark
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Anderson
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Bonomo
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D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015151:Immunoblotting; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*; D047090:beta-Lactams / Q000378:metabolism*
D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases