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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 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:
; ; ; ; ; ; ; ;
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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Works referencing / citing this record:

Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules
journal, April 2017


Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis
journal, January 2017


The structure of Toho1 β-lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition
journal, November 2016


Crystal Structure of Phosphoserine BlaC from Mycobacterium tuberculosis Inactivated by Bis(Benzoyl) Phosphate
journal, July 2019


Application of profile fitting method to neutron time-of-flight protein single crystal diffraction data collected at the iBIX
journal, December 2016


Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam
journal, June 2019


Mechanisms of proton relay and product release by Class A β‐lactamase at ultrahigh resolution
journal, November 2017


Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate
journal, October 2016


The rise of neutron cryo-crystallography
journal, July 2018


The structure of Toho1 β-lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition
journal, November 2016


Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate
journal, October 2016


Crystal Structure of Phosphoserine BlaC from Mycobacterium tuberculosis Inactivated by Bis(Benzoyl) Phosphate
journal, July 2019