Mechanistic Roles of Conserved Active-Site Residues in OXA-48 Carbapenem Hydrolysis.

Abstract

Carbapenem-hydrolyzing class D β-lactamases are a major source of resistance to last resort β-lactam antibiotics. OXA-48 is a class D β-lactamase that hydrolyzes carbapenems through a catalytic mechanism involving a nucleophilic serine and a carboxylated lysine general base. To define the contributions of conserved active-site residues to catalysis and substrate turnover, alanine substitutions were introduced at key active site positions, followed by steady-state and presteady-state kinetic analysis of mutant enzymes for imipenem and cephalothin hydrolysis. Mutations produced distinct effects, revealing roles in substrate acylation and deacylation reactions. Substitutions at Val120, Trp157, and Leu158 cause the largest decreases in kcat for both imipenem and cephalothin hydrolysis. It is known that deacylation is the rate-limiting step in carbapenem hydrolysis by OXA-48 and presteady-state kinetics analysis shows that the V120A and L158A substitutions further slow the deacylation reaction for imipenem, reducing kcat and kcat/KM values. Our data are also consistent with acylation being rate-limiting for cephalothin hydrolysis, with the V120A and L158A substitutions reducing substrate binding affinity and/or the acylation rate. Structural analysis indicates that the V120A enzyme has open water channel to the noncarboxylated Lys73 while the L158A enzyme is also open to water but has a carboxylated Lys73. Together, these results support a model in which conserved hydrophobic active-site residues optimize the geometric and electrostatic environment required for efficient carbapenem deacylation and turnover. These findings provide mechanistic insight into OXA-48 catalysis that may be exploited to design inhibitors targeting the enzyme.