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 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 »
- Authors:
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1251612
- DOE Contract Number:
- AC02-06CH11357
- Resource Type:
- Journal Article
- 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)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
Citation Formats
Vandavasi, Venu Gopal, Weiss, Kevin L., Cooper, Jonathan B., Erskine, Peter T., Tomanicek, Stephen J., Ostermann, Andreas, Schrader, Tobias E., Ginell, Stephan L., and Coates, Leighton. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography. United States: N. p., 2016.
Web. doi:10.1021/acs.jmedchem.5b01215.
Vandavasi, Venu Gopal, Weiss, Kevin L., Cooper, Jonathan B., Erskine, Peter T., Tomanicek, Stephen J., Ostermann, Andreas, Schrader, Tobias E., Ginell, Stephan L., & Coates, Leighton. Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography. United States. https://doi.org/10.1021/acs.jmedchem.5b01215
Vandavasi, Venu Gopal, Weiss, Kevin L., Cooper, Jonathan B., Erskine, Peter T., Tomanicek, Stephen J., Ostermann, Andreas, Schrader, Tobias E., Ginell, Stephan L., and Coates, Leighton. 2016.
"Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography". United States. https://doi.org/10.1021/acs.jmedchem.5b01215.
@article{osti_1251612,
title = {Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography},
author = {Vandavasi, Venu Gopal and Weiss, Kevin L. and Cooper, Jonathan B. and Erskine, Peter T. and Tomanicek, Stephen J. and Ostermann, Andreas and Schrader, Tobias E. and Ginell, Stephan L. and Coates, Leighton},
abstractNote = {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 role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzymes native machinery.},
doi = {10.1021/acs.jmedchem.5b01215},
url = {https://www.osti.gov/biblio/1251612},
journal = {Journal of Medicinal Chemistry},
issn = {0022-2623},
number = 1,
volume = 59,
place = {United States},
year = {Thu Jan 14 00:00:00 EST 2016},
month = {Thu Jan 14 00:00:00 EST 2016}
}
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