OHIO-1 BETA-LACTAMASE RESISTANT TO MECHANISM-BASED INACTIVATORS

Spontaneous mutants of OHIO-1 beta-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100-mu-g/ml ampicillin plus 32-mu-g/ml clavulanic acid compared to less-than-or-equal-to 2-mu-g/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V(max)/K(m) ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K(i) for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18-mu-M, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine69 --> isoleucine69 substitution accounting for the observed changes in substrate specificity.