The hydrolytic water molecule of Class A β-lactamase relies on the acyl-enzyme intermediate ES* for proper coordination and catalysis

Yunjiao He, Jinping Lei, Xuehua Pan, Xuhui Huang, Yanxiang Zhao

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14 Scopus citations


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.
Original languageEnglish (US)
JournalScientific Reports
Issue number1
StatePublished - Jun 23 2020
Externally publishedYes

Bibliographical note

KAUST Repository Item: Exported on 2022-06-14
Acknowledgements: This work was supported by the Research Grants Council GRF grants PolyU151052/16M and PolyU151015/17M, Research Grants Council CRF grant C5030-14E, Research Grants Council AoE grantAoE/M-09/12, Health and Medical Research Fund grant 14130502, Shenzhen Science and Technology Innovation Commission (JCYJ20170818104619974 and JCYJ20180306173813203) and the Research Committee of the Hong Kong Polytechnic University. We thank Shanghai Synchrotron Radiation Facility (SSRF) beam line BL17U for help with data collection. This research made use of the Protein Crystallography Facility in Hong Kong Polytechnic University and the resources of the Supercomputing Laboratory at King Abdullah University of Science and Technology (KAUST). YH and XP cloned and purified the protein, solved the structures, and carried out the in vitro biophysical and biochemical assays. JL and XH conducted theoretical computations. YZ supervised the project and wrote the manuscript. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interests.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.

ASJC Scopus subject areas

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