Capturing Catalysis: Structural Insights into the Acyl-Enzyme Intermediate of Priestia megaterium Penicillin G Acylase
Issued Date
2026-01-01
Resource Type
eISSN
21555435
Scopus ID
2-s2.0-105037817583
Journal Title
ACS Catalysis
Volume
16
Issue
9
Start Page
8146
End Page
8156
Rights Holder(s)
SCOPUS
Bibliographic Citation
ACS Catalysis Vol.16 No.9 (2026) , 8146-8156
Suggested Citation
Kaewsasan C., Rojviriya C., Oonanant W., Prathumrat N., Koinueng W., Yuvaniyama J. Capturing Catalysis: Structural Insights into the Acyl-Enzyme Intermediate of Priestia megaterium Penicillin G Acylase. ACS Catalysis Vol.16 No.9 (2026) , 8146-8156. 8156. doi:10.1021/acscatal.6c00015 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/116666
Title
Capturing Catalysis: Structural Insights into the Acyl-Enzyme Intermediate of Priestia megaterium Penicillin G Acylase
Corresponding Author(s)
Other Contributor(s)
Abstract
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.