OHIO-1 beta-lactamase mutants: Asp179Gly mutation confers resistance to ceftazidime

The Asp179Gly mutant of the OHIO-1 beta-lactamase, an SHV enzyme, was constructed to investigate the effect of disruption of the omega loop on beta-lactamase activity in this class A enzyme. In Escherichia coli DH5 alpha the strain possessing the Asp179Gly mutation of the OHIO-1 beta-lactamase demonstrated increased susceptibility to all beta-lactams except ceftazidime and ceftriaxone. The minimum inhibitory concentrations for ceftazidime and ceftriaxone increased from 0.25 and 0.015 mu g/ml to 4.0 and .25 mu g/ml respectively, For ceftazidime, a substrate not hydrolyzed by the wild-type enzyme (K-m greater than or equal to 500 mu M), the K-m of the Asp179Gly mutant beta-lactamase was measured to be 7 mu M and the V-max was 0.13 mu M/min. The minimum inhibitory concentrations, K-m, and V-max for all other beta-lactams decreased. Our analysis of this OHIO-1 beta-lactamase mutant suggests that disruption of the salt bridge in the omega loop by substitution of a glycine at position 179 markedly decreases the catalytic efficiency of the enzyme.