Journal
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
Volume 61, Issue 1, Pages -Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AAC.01636-16
Keywords
beta-lactamase; aztreonam; acyl-enzyme complex; neutron structure; X-ray structure
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Funding
- Laboratory Directed Research and Development Program at Oak Ridge National Laboratory (ORNL)
- Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy
- Oak Ridge National Laboratory's Center for Structural Molecular Biology (CSMB)
- U.S. Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
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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 beta-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M beta-lactamase but not 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 beta-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A beta-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found 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, as previously proposed.
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