title: A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations.
abstract: Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
authors.forename: Alan-Shaun
authors.initials: AS
authors.lastname: Wilkinson
authors.forename: Patrick K
authors.initials: PK
authors.lastname: Bryant
authors.forename: Samy O
authors.initials: SO
authors.lastname: Meroueh
authors.forename: Malcolm G P
authors.initials: MG
authors.lastname: Page
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Wharton
mesh_terms: D000215:Acylation; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000145:classification; D001398:Aztreonam / Q000737:chemistry*; D001665:Binding Sites; D016971:Citrobacter freundii / Q000201:enzymology*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004952:Esters; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008712:Methicillin / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D004952:Esters; D001618:beta-Lactamases; D001398:Aztreonam; D008712:Methicillin