Capturing Catalysis: Structural Insights into the Acyl-Enzyme Intermediate of <i>Priestia megaterium</i> Penicillin G Acylase

Penicillin G acylase (PGA) is a key biocatalyst in the synthesis of semisynthetic β-lactam antibiotics. We herein report three high-resolution crystal structures of Priestia megaterium PGA (PmPGA), capturing the enzyme in distinct catalytic states: the ligand-free form, the enzyme–product complex (PmPGA–PAA), and the covalent acyl-enzyme intermediate (PmPGA–PAX). These structures provide direct structural evidence for the proposed two-step catalytic mechanism and offer insights into the roles of active-site residues and water molecules in catalysis. The nucleophilic Ser1β, whose catalytic role is well established, is activated by its α-amino group and stabilized by Gln23β and Asn245β, while Ala69β and Asn245β form the oxyanion hole. Water molecules at the Wat1 position appear to mediate proton transfer and nucleophilic attack during acylation and deacylation, respectively. Structural comparisons with Escherichia coli PGA (EcPGA) highlight both conserved features and adaptations in PmPGA, including a shorter active-site loop and a second calcium-binding site. Notably, PmPGA selectively recognizes the side chain of its substrate rather than the β-lactam core, suggesting a distinct substrate specificity that can be leveraged for the design of tailored biocatalysts. These findings deepen our understanding of Ntn hydrolase catalysis and establish PmPGA as a promising scaffold for engineering next-generation enzymes for β-lactam antibiotic synthesis.