Construction and characterization of an OHIO-1 beta-lactamase bearing Met69Ile and Gly238Ser mutations

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Abstract
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 Km values of cefotaxime and ceftazidime for OHIO-1 and Met69Ile beta-lactamases were > or = 500 microM. The Km of cefotaxime for the Gly238Ser beta-lactamase was 26 microM, and that of ceftazidime was 105 microM. The Km of cefotaxime for the Met69Ile-Gly238Ser beta-lactamase was 292 microM, and that of ceftazidime was 392 microM. For the beta-lactamase inhibitors clavulanate and sulbactam, the apparent Ki values for the Met69Ile-Gly238Ser enzyme were 0.03 and 0.15 microM, respectively. Relative Vmax 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 DH5alpha 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.

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