title: Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.
abstract: The catalytic mechanism of class A β-lactamases is often debated due in part to the large number of amino acids that interact with bound β-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type β-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 β-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.
authors.affiliation: Oak Ridge National Laboratory , 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States.
authors.forename: Venu Gopal
authors.initials: VG
authors.lastname: Vandavasi
authors.affiliation: Oak Ridge National Laboratory , 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Weiss
authors.affiliation: Birkbeck University of London , Malet Street, London WC1E 7HX, United Kingdom.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cooper
authors.affiliation: Birkbeck University of London , Malet Street, London WC1E 7HX, United Kingdom.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Erskine
authors.affiliation: Oak Ridge National Laboratory , 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tomanicek
authors.affiliation: Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München , Lichtenbergstr. 1, 85748 Garching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.affiliation: Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH , Lichtenbergstr. 1, 85747 Garching, Germany.
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.affiliation: Structural Biology Center, Argonne National Laboratory , 9700 St. Cass Avenue, Argonne, Illinois 60439, United States.
authors.forename: Stephan L
authors.initials: SL
authors.lastname: Ginell
authors.affiliation: Oak Ridge National Laboratory , 1 Bethel Valley Road, Oak Ridge, Tennessee 37831, United States.
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
mesh_terms: D000215:Acylation; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002439:Cefotaxime / Q000378:metabolism; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D033363:Neutron Diffraction; D011487:Protein Conformation; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D047090:beta-Lactams; D001618:beta-Lactamases; D002439:Cefotaxime