The role of OXA-1 β-lactamase Asp⁶⁶ in the stabilization of the active-site carbamate group and in substrate turnover

The OXA-1 β-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser⁶⁷. In class A β-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp⁶⁶ in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp⁶⁶ mutants expressed in Escherichia coli DH10B ranged from ≤8 μg/ml for cysteine, proline and the basic amino acids to ≥256 μg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys⁷⁰, that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria–Bertani-broth agar resulted in a ≥16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp⁶⁶ forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO₂ bound to Lys⁷⁰ and thereby profoundly affects substrate turnover.