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 K-m values of cefotaxime and ceftazidime for OHIO-1 and Met69Ile beta-lactamases were greater than or equal to 500 mu M. The K-m of cefotaxime for the Gly238Ser beta-lactamase was 26 mu M, and that of ceftazidime was 105 mu M. The K-m of cefotaxime for the Met69Ile-Gly238Ser beta-lactamase was 292 mu M, and that of ceftazidime was 392 mu M, For the beta-lactamase inhibitors clavulanate and sulbactam, the apparent K-i values for the Met69Ile-Gly238Ser enzyme were 0.03 and 0.15 mu M, respectively, Relative V-max 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 DH5 alpha 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.