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. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-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 β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-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.
authors: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors: Venu Gopal
authors: VG
authors: Vandavasi
authors: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors: Patricia S
authors: 0000-0002-1097-5958
authors: PS
authors: Langan
authors: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors: Kevin L
authors: 0000-0002-6486-8007
authors: KL
authors: Weiss
authors: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors: Jerry M
authors: 0000-0002-3103-9333
authors: JM
authors: Parks
authors: Birkbeck University of London, London, United Kingdom.
authors: Jonathan B
authors: JB
authors: Cooper
authors: Structural Biology Center, Argonne National Laboratory, Argonne, Illinois, USA.
authors: Stephan L
authors: SL
authors: Ginell
authors: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA coatesl@ornl.gov.
authors: Leighton
authors: 0000-0003-2342-049X
authors: L
authors: Coates
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001398:Aztreonam / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008826:Microbial Sensitivity Tests; D008997:Monobactams / Q000737:chemistry / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D008997:Monobactams; D001618:beta-Lactamases; D001398:Aztreonam
keywords: X-ray structure; acyl-enzyme complex; aztreonam; neutron structure; β-lactamase