Abstract
Serine-based β-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate β-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the β-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated β-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A β-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi–Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent β-lactamase-mediated antibiotic resistance.
Highlights
Over 1,000 β-lactamases have been reported so far and they are grouped into four classes A to D based on sequence and structural similarities[1,2]
Two general base residues are required in this process, one to activate the catalytic serine residue in the acylation step, and the other to activate the hydrolytic water molecule in the deacylation step
Our crystallographic studies of Glu166Tyr led to identification of previously underappreciated structural and environmental factors in the active site that work synergistically to coordinate and activate the hydrolytic water molecule for nucleophilic attack on ES*
Summary
Over 1,000 β-lactamases have been reported so far and they are grouped into four classes A to D based on sequence and structural similarities[1,2]. In Class A β-lactamases, Ser[70] is the key catalytic residue while Lys[73] and Glu[166] serve as the general base for acylation and deacylation steps respectively (Fig. 1a)[6].
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