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This content will become publicly available on December 2, 2016

Title: Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography

The catalytic mechanism of class A beta-lactamases is often debated due in part to the large number of amino acids that interact with bound beta-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type beta-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 beta-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 rolemore » of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.« less
Authors:
 [1] ;  [1] ;  [2] ;  [2] ;  [1] ;  [3] ;  [4] ;  [5] ;  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Birkbeck Univ., London (United Kingdom)
  3. Technical Univ. of Munchen, Garching (Germany)
  4. Julich Center for Neutron Science (JCNS), Garching (Germany)
  5. Argonne National Lab. (ANL), Argonne, IL (United States)
Publication Date:
OSTI Identifier:
1261502
Grant/Contract Number:
AC05-00OR22725; AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Journal of Medicinal Chemistry
Additional Journal Information:
Journal Volume: 59; Journal Issue: 1; Journal ID: ISSN 0022-2623
Publisher:
American Chemical Society (ACS)
Research Org:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source; Center for Structural Molecular Biology (CSMB)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND TECHNOLOGY; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY