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

Abstract

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:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1393811
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article: 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
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Cooper, Jonathan B., Weiss, Kevin L., Coates, Leighton, Parks, Jerry M., and Vandavasi, Venu Gopal. Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate. United States: N. p., 2016. Web. doi:10.1128/AAC.01636-16.
Cooper, Jonathan B., Weiss, Kevin L., Coates, Leighton, Parks, Jerry M., & Vandavasi, Venu Gopal. Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate. United States. doi:10.1128/AAC.01636-16.
Cooper, Jonathan B., Weiss, Kevin L., Coates, Leighton, Parks, Jerry M., and Vandavasi, Venu Gopal. Mon . "Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate". United States. doi:10.1128/AAC.01636-16. https://www.osti.gov/servlets/purl/1393811.
@article{osti_1393811,
title = {Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate},
author = {Cooper, Jonathan B. and Weiss, Kevin L. and Coates, Leighton and Parks, Jerry M. and Vandavasi, Venu Gopal},
abstractNote = {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.},
doi = {10.1128/AAC.01636-16},
journal = {Antimicrobial Agents and Chemotherapy},
number = 1,
volume = 61,
place = {United States},
year = {Mon Oct 24 00:00:00 EDT 2016},
month = {Mon Oct 24 00:00:00 EDT 2016}
}

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Cited by: 6 works
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Works referenced in this record:

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

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