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Title: Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.
Authors:
 [1] ; ORCiD logo [2] ; ORCiD logo [2] ; ORCiD logo [2] ;  [2]
  1. Univ. of Southampton (United Kingdom)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Antimicrobial Agents and Chemotherapy
Additional Journal Information:
Journal Volume: 61; Journal Issue: 1; Journal ID: ISSN 0066-4804
Publisher:
American Society for Microbiology
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1393811