Defining the role of Asp66 in the Class D β‐lactamase OXA‐1

Abstract

The β-lactamase OXA-1 is the only class D enzyme that has an aspartate at residue 66, a position that is proximal to the critical nucleophillic Ser67 and helps form the floor of the active site. Residues adjacent to the active site serine have been shown to play a crucial role in inhibitor resistance in Class A enzymes such as TEM and SHV. To probe the influence of Asp66 on substrate affinity, we performed site-saturation mutagenesis at this position. Ampicillin MIC values for the full set of Asp66 mutants ranged from ≤ 8 μ g/ml for Cys, Pro and the basic amino acids, to ≥ 256 μ g/ml for Asn, Leu and the wild-type Asp. Substitution of Asn for Asp at position 66 also led to a moderate enhancement of third-generation cephalosporin resistance. This latter result is explained by a lower cefotaxime Km for Asp66Asn (14 μM) compared to wild-type (23 μM). OXA-1 shares with its fellow Class D enzymes a carbamylated Lys70 that acts as a general base in the catalysis of penicillins. The addition of 25 mM bicarbonate to LB agar resulted in a greater than 2 fold increase in MICs for most amino acid substitutions at position 66, but had little effect on resistance conferred by wild-type OXA-1 in E. coli DH10B. Because Asp66 forms hydrogen bonds with several other active site residues in the wild-type enzyme, the data suggest that Asp66 may play a role in stabilizing the CO2 bound to Lys70 and profoundly affect substrate turnover.