National Center for
5KSC: E166A/R274N/R276N Toho-1 Beta-lactamase aztreonam acyl-enzyme intermediate
Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A beta-Lactamase with a Monobactam Substrate
Antimicrob. Agents Chemother. (2016) 61
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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