Perturbing the General Base Residue Glu166 in the Active Site of Class A β-Lactamase Leads to Enhanced Carbapenem Binding and Acylation

Most class A β-lactamases cannot hydrolyze carbapenem antibiotics effectively. The molecular mechanism of this catalytic inefficiency has been attributed to the unique stereochemistry of carbapenems, including their 6-α-hydroxyethyl side chain and the transition between two tautomeric states when bound at the active site. Previous studies have shown that the 6-α-hydroxyethyl side chain of carbapenems can interfere with catalysis by forming hydrogen bonds with the deacylation water molecule to reduce its nucleophilicity. Here our studies of a class A noncarbapenemase PenP demonstrate that substituting the general base residue Glu166 with Ser or other residues leads to a significant enhancement of the acylation kinetics by ∼100–500 times toward carbapenems like meropenem. The structures of PenP and Glu166Ser both in apo form and in complex with meropenem reveal that Glu166 is critical for the formation of a hydrogen bonding network within the active site that locks Asn170 in an orientation to impose steric ...