skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis

Abstract

Here, the emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of β-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and pKas of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with Nζ of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 ismore » activated for nucleophilic attack of the β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the acyl-enzyme intermediate.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [1];  [3]; ORCiD logo [4]; ORCiD logo [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Univ. of Tennessee, Knoxville, TN (United States)
  3. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. at Buffalo, Buffalo, NY (United States)
  4. Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center
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); National Science Foundation (NSF)
OSTI Identifier:
1459304
Alternate Identifier(s):
OSTI ID: 1485285
Grant/Contract Number:  
AC05-00OR22725; AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
ACS Catalysis
Additional Journal Information:
Journal Volume: 8; Journal Issue: 3; Journal ID: ISSN 2155-5435
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; antibiotic resistance; antibiotics; crystallography; enzyme; enzyme structure; simulation

Citation Formats

Langan, Patricia S., Vandavasi, Venu Gopal, Cooper, Sarah J., Weiss, Kevin L., Ginell, Stephan L., Parks, Jerry M., and Coates, Leighton. Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis. United States: N. p., 2018. Web. doi:10.1021/acscatal.7b04114.
Langan, Patricia S., Vandavasi, Venu Gopal, Cooper, Sarah J., Weiss, Kevin L., Ginell, Stephan L., Parks, Jerry M., & Coates, Leighton. Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis. United States. doi:10.1021/acscatal.7b04114.
Langan, Patricia S., Vandavasi, Venu Gopal, Cooper, Sarah J., Weiss, Kevin L., Ginell, Stephan L., Parks, Jerry M., and Coates, Leighton. Thu . "Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis". United States. doi:10.1021/acscatal.7b04114. https://www.osti.gov/servlets/purl/1459304.
@article{osti_1459304,
title = {Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis},
author = {Langan, Patricia S. and Vandavasi, Venu Gopal and Cooper, Sarah J. and Weiss, Kevin L. and Ginell, Stephan L. and Parks, Jerry M. and Coates, Leighton},
abstractNote = {Here, the emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of β-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and pKas of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with Nζ of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 is activated for nucleophilic attack of the β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the acyl-enzyme intermediate.},
doi = {10.1021/acscatal.7b04114},
journal = {ACS Catalysis},
number = 3,
volume = 8,
place = {United States},
year = {2018},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 11 works
Citation information provided by
Web of Science

Figures / Tables:

Figure 1 Figure 1: Catalytic mechanism of class A β-lactamase inactivation of a β-lactam substrate. A serine nucleophile cleaves the β-lactam bond of the substrate in two steps, acylation and deacylation, which lead to hydrolysis: First, the pre-covalent enzyme-substrate complex is formed and the acylation reaction is initiated (1). General base-catalyzed nucleophilicmore » attack on the β-lactam carbonyl by the serine hydroxy group proceeds through a tetrahedral intermediate (2) and forms a transient acyl-enzyme adduct (3). The acyl-enzyme adduct (3) undergoes general base-catalyzed attack by the hydrolytic water molecule and forms a second tetrahedral intermediate during deacylation (4), which subsequently collapses to form a post-covalent complex (5) prior to release of the hydrolyzed product.« less

Save / Share:
Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.