title: Neutron and X-ray crystal structures of a perdeuterated enzyme inhibitor complex reveal the catalytic proton network of the Toho-1 β-lactamase for the acylation reaction.
abstract: The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with (11)B, as (10)B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction.
authors.affiliation: Oak Ridge National Laboratory, OakRidge, Tennessee 37831, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tomanicek
authors.forename: Robert F
authors.initials: RF
authors.lastname: Standaert
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Weiss
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Ng
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
mesh_terms: D000215:Acylation; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D008024:Ligands; D008956:Models, Chemical; D008968:Molecular Conformation; D009502:Neutrons; D009584:Nitrogen / Q000737:chemistry; D011522:Protons; D013876:Thiophenes / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D008024:Ligands; D011522:Protons; D013876:Thiophenes; C088015:benzothiophene; D006859:Hydrogen; D001618:beta-Lactamases; C495977:beta-lactamase Toho-1, E coli; D009584:Nitrogen