5ZFT

Crystal structure of beta-lactamase PenP mutant-E166Y in complex with cephaloridine as "pre-deacylation" intermediate

Summary for 5ZFT

• Entry DOI: 10.2210/pdb5zft/pdb
• Descriptor: Beta-lactamase, 5-METHYL-2-[2-OXO-1-(2-THIOPHEN-2-YL-ACETYLAMINO)-ETHYL]-3,6-DIHYDRO-2H-[1,3]THIAZINE-4-CARBOXYLIC ACID (3 entities in total)
• Functional Keywords: class a beta-lactamase, antibiotic resistance, hydrolase
• Biological source: Bacillus licheniformis
• Total number of polymer chains: 2
• Total formula weight: 60404.55

Authors

Pan, X.,Zhao, Y. (deposition date: 2018-03-07, release date: 2019-03-20, Last modification date: 2023-11-22)

Primary citation

He, Y.,Lei, J.,Pan, X.,Huang, X.,Zhao, Y.
The hydrolytic water molecule of Class A beta-lactamase relies on the acyl-enzyme intermediate ES* for proper coordination and catalysis.
Sci Rep, 10:10205-10205, 2020

PubMed 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.

PubMed: 32576842
DOI: 10.1038/s41598-020-66431-w

Experimental method

X-RAY DIFFRACTION (1.93 Å)