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

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

Journal Article · · Journal of Medicinal Chemistry
; ; ; ; ; ; ; ;

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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)
DOE Contract Number:
AC02-06CH11357
OSTI ID:
1251612
Journal Information:
Journal of Medicinal Chemistry, Vol. 59, Issue 1; ISSN 0022-2623
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English

References (34)

A standard numbering scheme for the class A β-lactamases journal May 1991
Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability journal April 2005
The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity journal January 1999
Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure journal December 1995
Growing Group of Extended-Spectrum  -Lactamases: the CTX-M Enzymes journal December 2003
Catalytic properties of class A β-lactamases: efficiency and diversity journal March 1998
CTX-M-type β-lactamases: an emerging group of extended-spectrum enzymes journal March 2000
TEM- and SHV-derived extended-spectrum β-lactamases: relationship between selection, structure and function journal January 1995
Crystal structure of the E166A mutant of extended-spectrum β-lactamase toho-1 at 1.8 Å resolution 1 1Edited by R. Huber journal February 1999
Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli journal October 1995
The Acylation Mechanism of CTX-M β-Lactamase at 0.88 Å Resolution journal May 2007
An Ultrahigh Resolution Structure of TEM-1 β-Lactamase Suggests a Role for Glu166 as the General Base in Acylation journal May 2002
Site-Directed Mutagenesis of Glutamate-166 in .beta.-Lactamase Leads to a Branched Path Mechanism journal June 1994
Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution journal May 1987
Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution journal October 1992
Ab Initio QM/MM Study of Class A β-Lactamase Acylation:  Dual Participation of Glu166 and Lys73 in a Concerted Base Promotion of Ser70 journal November 2005
Site-directed Mutagenesis of Glutamate 166 in Two β-Lactamases: KINETIC AND MOLECULAR MODELING STUDIES journal February 1997
A catalytically-impaired class A β -lactamase: 2 Å crystal structure and kinetics of the Bacillus licheniformis E166A mutant journal January 1993
The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins journal May 2004
Crystal Structure of an Acylation Transition-State Analog of the TEM-1 β-Lactamase. Mechanistic Implications for Class A β-Lactamases † journal February 1998
Improvement of crystal quality by surface mutations of β-lactamase Toho-1 journal March 2009
The catalytic efficiency (kcat/Km) of the class A β-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog journal April 2010
Neutron macromolecular crystallography journal June 2009
Deuterium Labeling for Neutron Structure-Function-Dynamics Analysis book January 2009
The macromolecular neutron diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge: enhanced optics design, high-resolution neutron detectors and simulated diffraction journal April 2010
Joint X-ray and neutron refinement with phenix.refine journal October 2010
Cryogenic neutron protein crystallography: routine methods and potential benefits journal June 2014
The active site protonation states of perdeuterated Toho-1 β-lactamase determined by neutron diffraction support a role for Glu166 as the general base in acylation journal December 2010
Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin journal September 2002
Structure, Function, and Inhibition along the Reaction Coordinate of CTX-M β-Lactamases journal April 2005
Crystal Structure of Extended-Spectrum β-Lactamase Toho-1:  Insights into the Molecular Mechanism for Catalytic Reaction and Substrate Specificity Expansion † , ‡ journal September 2003
Mechanism of acyl transfer by the class A serine β-lactamase of Streptomyces albus G journal October 1991
Protonation of the  -lactam nitrogen is the trigger event in the catalytic action of class A  -lactamases journal March 2000
Structural Consequences of the Inhibitor-Resistant Ser130Gly Substitution in TEM β-Lactamase † , ‡ journal July 2005

Cited By (12)

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
In vivo functional analysis of a class A β-lactamase-related protein essential for clavulanic acid biosynthesis in Streptomyces clavuligerus journal April 2019
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
Dynamical Behavior of β-Lactamases and Penicillin- Binding Proteins in Different Functional States and Its Potential Role in Evolution journal November 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
Structural/mechanistic insights into the efficacy of nonclassical β-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates: β-lactamase inhibitors versus S. maltophilia journal September 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

Similar Records

Exploring the Mechanism of β-Lactam Ring Protonation in the Class A β-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography
Journal Article · Wed Dec 02 00:00:00 EST 2015 · Journal of Medicinal Chemistry · OSTI ID:1251612
+6 more
Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis
Journal Article · Thu Feb 15 00:00:00 EST 2018 · ACS Catalysis · OSTI ID:1251612
+4 more
Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution
Journal Article · Mon Nov 20 00:00:00 EST 2017 · Federation of European Biochemical Societies (FEBS) Journal · OSTI ID:1251612
+3 more

Related Subjects

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY