OHIO-1 β-lactamase mutants: the Arg 244Ser mutant and resistance to β-lactams and β-lactamase inhibitors

Abstract

Mutations at residue 244 (Ambler numbering system) in the class A TEM β-lactamase confer resistance to inactivation by β-lactamase inhibitors and result in diminished turnover of β-lactam substrates. The Arg 244Ser mutant of the OHIO-1 β-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to β-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5α expressing the Arg 244Ser β-lactamase were reduced when compared to the strain bearing the OHIO-1 β-lactamase: ampicillin, 512 vs. 8192 μg ml −1; cephaloridine, 4 vs. 32 μg ml −1, respectively. The MICs for the β-lactam β-lactamase inhibitor combinations demonstrated resistance only to ampicillin–clavulanate, 16/8 vs. 8/4 μg ml −1 respectively. In contrast, there was increased susceptibility to ampicillin–sulbactam, ampicillin–tazobactam, and piperacillin–tazobactam. When compared to the OHIO-1 β-lactamase homogenous preparations of the Arg 244Ser β-lactamase enzyme demonstrated increased K m and decreased k cat values for benzylpenicillin ( K m=17 vs. 50 μM, k cat=345 vs. 234 s −1) and cephaloridine ( K m=97 vs. 202 μM, k cat=1023 vs. 202 s −1). Although the K i and IC 50 values were increased for each inhibitor when compared to OHIO-1 β-lactamase, the turnover numbers ( t n) required for inactivation were increased only for clavulanate. For the Arg 244Ser mutant enzyme of OHIO-1, the increased K i, decreased t n for the sulfones, and different partition ratio ( k cat/ k inact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A β-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.