Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

Judge, Allison; Hu, Liya; Sankaran, Banumathi; Van Riper, Justin; Venkataram Prasad, B. V.; Palzkill, Timothy

doi:10.1038/s42003-023-04422-z

Title: Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

Journal Article · Thu Jan 12 00:00:00 EST 2023 · Communications Biology

DOI:https://doi.org/10.1038/s42003-023-04422-z· OSTI ID:1909043

; Hu, Liya;

; Van Riper, Justin;

;

Abstract CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.

View Journal Article

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH); Welch Foundation

Grant/Contract Number:: AC02-05CH11231; AI32956; Q1279; T32 GM120011; P30 GM124169-01

OSTI ID:: 1909043

Alternate ID(s):: OSTI ID: 1972190

Journal Information:: Communications Biology, Journal Name: Communications Biology Vol. 6 Journal Issue: 1; ISSN 2399-3642

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (60)

In Vitro Selection of Meropenem Resistance among Ceftazidime-Avibactam-Resistant, Meropenem-Susceptible Klebsiella pneumoniae Isolates with Variant KPC-3 Carbapenemases Shields, Ryan K.; Nguyen, M. Hong; Press, Ellen G. Antimicrobial Agents and Chemotherapy, Vol. 61, Issue 5 https://doi.org/10.1128/AAC.00079-17	journal	May 2017
ENDOR Structural Characterization of a Catalytically Competent Acylenzyme Reaction Intermediate of Wild-Type TEM-1 β-Lactamase Confirms Glutamate-166 as the Base Catalyst Mustafi, Devkumar; Sosa-Peinado, Alejandro; Makinen, Marvin W. Biochemistry, Vol. 40, Issue 8 https://doi.org/10.1021/bi0021075	journal	January 2001
Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β -lactamase studied by site-directed mutagenesis Jacob, F.; Joris, B.; Lepage, S. Biochemical Journal, Vol. 271, Issue 2 https://doi.org/10.1042/bj2710399	journal	October 1990
Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability Chen, Yu; Delmas, Julien; Sirot, Jacques Journal of Molecular Biology, Vol. 348, Issue 2 https://doi.org/10.1016/j.jmb.2005.02.010	journal	April 2005
Overview of the CCP 4 suite and current developments Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910045749	journal	March 2011
Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G. Biochemistry, Vol. 54, Issue 2 https://doi.org/10.1021/bi501195g	journal	December 2014
Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis Brown, Nicholas G.; Shanker, Sreejesh; Prasad, B. V. Venkataram Journal of Biological Chemistry, Vol. 284, Issue 48 https://doi.org/10.1074/jbc.M109.053819	journal	October 2009
Deep Sequencing of Systematic Combinatorial Libraries Reveals β-Lactamase Sequence Constraints at High Resolution Deng, Zhifeng; Huang, Wanzhi; Bakkalbasi, Erol Journal of Molecular Biology, Vol. 424, Issue 3-4 https://doi.org/10.1016/j.jmb.2012.09.014	journal	December 2012
Towards automated crystallographic structure refinement with phenix.refine Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4 https://doi.org/10.1107/S0907444912001308	journal	March 2012
Trimmomatic: a flexible trimmer for Illumina sequence data Bolger, Anthony M.; Lohse, Marc; Usadel, Bjoern Bioinformatics, Vol. 30, Issue 15 https://doi.org/10.1093/bioinformatics/btu170	journal	April 2014
Three Decades of -Lactamase Inhibitors Drawz, S. M.; Bonomo, R. A. Clinical Microbiology Reviews, Vol. 23, Issue 1 https://doi.org/10.1128/CMR.00037-09	journal	January 2010
Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability Mehta, Shrenik C.; Rice, Kacie; Palzkill, Timothy PLOS Pathogens, Vol. 11, Issue 6 https://doi.org/10.1371/journal.ppat.1004949	journal	June 2015
Mechanisms of proton relay and product release by Class A β‐lactamase at ultrahigh resolution Lewandowski, Eric M.; Lethbridge, Kathryn G.; Sanishvili, Ruslan The FEBS Journal, Vol. 285, Issue 1 https://doi.org/10.1111/febs.14315	journal	November 2017
Unravelling ceftazidime/avibactam resistance of KPC-28, a KPC-2 variant lacking carbapenemase activity Oueslati, Saoussen; Iorga, Bogdan I.; Tlili, Linda Journal of Antimicrobial Chemotherapy, Vol. 74, Issue 8 https://doi.org/10.1093/jac/dkz209	journal	May 2019
Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases Patel, Meha P.; Fryszczyn, Bartlomiej G.; Palzkill, Timothy Antimicrobial Agents and Chemotherapy, Vol. 59, Issue 11 https://doi.org/10.1128/AAC.00618-15	journal	August 2015
Klebsiella pneumoniae Carbapenemase Variants Resistant to Ceftazidime-Avibactam: an Evolutionary Overview Hobson, Claire Amaris; Pierrat, Gautier; Tenaillon, Olivier Antimicrobial Agents and Chemotherapy, Vol. 66, Issue 9 https://doi.org/10.1128/aac.00447-22	journal	September 2022
Effect of D240G substitution in a novel ESBL CTX-M-27 Bonnet, R. Journal of Antimicrobial Chemotherapy, Vol. 52, Issue 1 https://doi.org/10.1093/jac/dkg256	journal	June 2003
Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum β-Lactamase CTX-M-9 Delmas, J.; Robin, F.; Carvalho, F. Antimicrobial Agents and Chemotherapy, Vol. 50, Issue 2 https://doi.org/10.1128/AAC.50.2.731-738.2006	journal	February 2006
UCSF Chimera?A visualization system for exploratory research and analysis Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Journal of Computational Chemistry, Vol. 25, Issue 13 https://doi.org/10.1002/jcc.20084	journal	January 2004
The Structure of <latex>$\beta$</latex>-Lactamases Ambler, R. P. Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 289, Issue 1036 https://doi.org/10.1098/rstb.1980.0049	journal	May 1980
Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance Patel, Meha P.; Hu, Liya; Brown, Cameron A. Journal of Biological Chemistry, Vol. 293, Issue 46 https://doi.org/10.1074/jbc.RA118.003792	journal	October 2018
Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC β-lactamases Palzkill, Timothy Frontiers in Molecular Biosciences, Vol. 5 https://doi.org/10.3389/fmolb.2018.00016	journal	February 2018
Structure and Dynamics of CTX-M Enzymes Reveal Insights into Substrate Accommodation by Extended-spectrum β-Lactamases Delmas, Julien; Chen, Yu; Prati, Fabio Journal of Molecular Biology, Vol. 375, Issue 1 https://doi.org/10.1016/j.jmb.2007.10.026	journal	January 2008
Functional Characterization of CTX-M-14 and CTX-M-15 β-Lactamases by In Vitro DNA Shuffling Po, Kathy Hiu Laam; Chan, Edward Wai Chi; Chen, Sheng Antimicrobial Agents and Chemotherapy, Vol. 61, Issue 12 https://doi.org/10.1128/AAC.00891-17	journal	December 2017
Structure-function studies of arginine at position 276 in CTX-M -lactamases Perez-Llarena, F. J.; Cartelle, M.; Mallo, S. Journal of Antimicrobial Chemotherapy, Vol. 61, Issue 4 https://doi.org/10.1093/jac/dkn031	journal	February 2008
Structure and Kinetics of the β-Lactamase Mutants S70A and K73H from Staphylococcus aureus PC1 ^† ^, ^‡ Chen, Celia C.; Smith, Tom J.; Kapadia, Geeta Biochemistry, Vol. 35, Issue 38 https://doi.org/10.1021/bi961153v	journal	January 1996
iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM Battye, T. Geoff G.; Kontogiannis, Luke; Johnson, Owen Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444910048675	journal	March 2011
A drug-resistant β-lactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency Soeung, Victoria; Lu, Shuo; Hu, Liya Journal of Biological Chemistry, Vol. 295, Issue 52 https://doi.org/10.1074/jbc.RA120.016103	journal	December 2020
Structural Basis of Extended Spectrum TEM β-Lactamases Maveyraud, Laurent; Saves, Isabelle; Burlet-Schiltz, Odile Journal of Biological Chemistry, Vol. 271, Issue 18 https://doi.org/10.1074/jbc.271.18.10482	journal	May 1996
Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis Kimura, Soichiro; Ishiguro, Masaji; Ishii, Yoshikazu Antimicrobial Agents and Chemotherapy, Vol. 48, Issue 5 https://doi.org/10.1128/AAC.48.5.1454-1460.2004	journal	May 2004
Contributions of Aspartate 49 and Phenylalanine 142 Residues of a Tight Binding Inhibitory Protein of β-Lactamases Petrosino, Joseph; Rudgers, Gary; Gilbert, Hiram Journal of Biological Chemistry, Vol. 274, Issue 4 https://doi.org/10.1074/jbc.274.4.2394	journal	January 1999
Polder maps: improving OMIT maps by excluding bulk solvent Liebschner, Dorothee; Afonine, Pavel V.; Moriarty, Nigel W. Acta Crystallographica Section D Structural Biology, Vol. 73, Issue 2, p. 148-157 https://doi.org/10.1107/S2059798316018210	journal	February 2017
Growing Group of Extended-Spectrum -Lactamases: the CTX-M Enzymes Bonnet, R. Antimicrobial Agents and Chemotherapy, Vol. 48, Issue 1 https://doi.org/10.1128/AAC.48.1.1-14.2004	journal	December 2003
An Ultrahigh Resolution Structure of TEM-1 β-Lactamase Suggests a Role for Glu166 as the General Base in Acylation Minasov, George; Wang, Xiaojun; Shoichet, Brian K. Journal of the American Chemical Society, Vol. 124, Issue 19 https://doi.org/10.1021/ja0259640	journal	May 2002
Distant and New Mutations in CTX-M-1 β-Lactamase Affect Cefotaxime Hydrolysis Pérez-Llarena, Francisco José; Kerff, Frédéric; Abián, Olga Antimicrobial Agents and Chemotherapy, Vol. 55, Issue 9 https://doi.org/10.1128/AAC.00298-11	journal	July 2011
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
REFMAC 5 for the refinement of macromolecular crystal structures Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911001314	journal	March 2011
The Acylation Mechanism of CTX-M β-Lactamase at 0.88 Å Resolution Chen, Yu; Bonnet, Richard; Shoichet, Brian K. Journal of the American Chemical Society, Vol. 129, Issue 17 https://doi.org/10.1021/ja0712064	journal	May 2007
The hydrolytic water molecule of Class A β-lactamase relies on the acyl-enzyme intermediate ES* for proper coordination and catalysis He, Yunjiao; Lei, Jinping; Pan, Xuehua Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-66431-w	journal	June 2020
Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes Delmas, Julien; Leyssene, David; Dubois, Damien Journal of Molecular Biology, Vol. 400, Issue 1 https://doi.org/10.1016/j.jmb.2010.04.062	journal	July 2010
Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution Strynadka, Natalie C. J.; Adachi, Hiroyuki; Jensen, Susan E. Nature, Vol. 359, Issue 6397 https://doi.org/10.1038/359700a0	journal	October 1992
An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M β-lactamase drug-resistance enzymes Lu, Shuo; Hu, Liya; Lin, Hanfeng Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-34564-3	journal	November 2022
The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko Biochemistry, Vol. 56, Issue 27 https://doi.org/10.1021/acs.biochem.7b00176	journal	June 2017
A Ceftazidime-Avibactam-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Strain Harboring bla _KPC-14 Isolated in New York City Niu, Siqiang; Chavda, Kalyan D.; Wei, Jie mSphere, Vol. 5, Issue 4 https://doi.org/10.1128/mSphere.00775-20	journal	August 2020
Extended-spectrum beta-lactamases Philippon, A.; Labia, R.; Jacoby, G. Antimicrobial Agents and Chemotherapy, Vol. 33, Issue 8 https://doi.org/10.1128/AAC.33.8.1131	journal	August 1989
Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin Shimamura, Tatsuro; Ibuka, Akiko; Fushinobu, Shinya Journal of Biological Chemistry, Vol. 277, Issue 48 https://doi.org/10.1074/jbc.M207884200	journal	September 2002
A standard numbering scheme for the class A β-lactamases Ambler, R. P.; Coulson, A. F. W.; Frère, J. M. Biochemical Journal, Vol. 276, Issue 1 https://doi.org/10.1042/bj2760269	journal	May 1991
Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations Sun, Zhizeng; Mehta, Shrenik C.; Adamski, Carolyn J. Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep33195	journal	September 2016
The Basis for Carbapenem Hydrolysis by Class A β-Lactamases: A Combined Investigation using Crystallography and Simulations Fonseca, Fátima; Chudyk, Ewa I.; van der Kamp, Marc W. Journal of the American Chemical Society, Vol. 134, Issue 44 https://doi.org/10.1021/ja304460j	journal	October 2012
Evolvability as a Function of Purifying Selection in TEM-1 β-Lactamase Stiffler, Michael A.; Hekstra, Doeke R.; Ranganathan, Rama Cell, Vol. 160, Issue 5 https://doi.org/10.1016/j.cell.2015.01.035	journal	February 2015
Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination Compain, Fabrice; Dorchène, Delphine; Arthur, Michel Antimicrobial Agents and Chemotherapy, Vol. 62, Issue 9 https://doi.org/10.1128/AAC.00357-18	journal	September 2018
Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase Doucet, Nicolas; De Wals, Pierre-Yves; Pelletier, Joelle N. Journal of Biological Chemistry, Vol. 279, Issue 44 https://doi.org/10.1074/jbc.M407606200	journal	August 2004
Mutation of the conserved Asp‐Asp pair impairs the structure, function, and inhibition of CTX‐M Class A β‐lactamase Kemp, M. Trent; Nichols, Derek A.; Zhang, Xiujun FEBS Letters, Vol. 595, Issue 24 https://doi.org/10.1002/1873-3468.14215	journal	November 2021
CTX-M-type β-lactamases: A successful story of antibiotic resistance D’Andrea, Marco Maria; Arena, Fabio; Pallecchi, Lucia International Journal of Medical Microbiology, Vol. 303, Issue 6-7 https://doi.org/10.1016/j.ijmm.2013.02.008	journal	August 2013
Mutational Events in Cefotaximase Extended-Spectrum β-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance Novais, Ângela; Cantón, Rafael; Coque, Teresa M. Antimicrobial Agents and Chemotherapy, Vol. 52, Issue 7 https://doi.org/10.1128/AAC.01658-07	journal	April 2008
Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography Nichols, Derek A.; Hargis, Jacqueline C.; Sanishvili, Ruslan Journal of the American Chemical Society, Vol. 137, Issue 25 https://doi.org/10.1021/jacs.5b00749	journal	June 2015
NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase Doucet, Nicolas; Savard, Pierre-Yves; Pelletier, Joelle N. Journal of Biological Chemistry, Vol. 282, Issue 29 https://doi.org/10.1074/jbc.M609777200	journal	July 2007
A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya Journal of Biological Chemistry, Vol. 290, Issue 16 https://doi.org/10.1074/jbc.M114.633438	journal	February 2015
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Information-theoretical entropy as a measure of sequence variability Shenkin, Peter S.; Erman, Batu; Mastrandrea, Lucy D. Proteins: Structure, Function, and Genetics, Vol. 11, Issue 4 https://doi.org/10.1002/prot.340110408	journal	December 1991

Similar Records

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

Journal Article · Wed Jun 14 00:00:00 EDT 2017 · Biochemistry · OSTI ID:1909043

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; +3 more

Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases

Journal Article · Tue Dec 09 00:00:00 EST 2014 · Biochemistry · OSTI ID:1909043

Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G.; +5 more

An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M β-lactamase drug-resistance enzymes

Journal Article · Mon Nov 07 00:00:00 EST 2022 · Nature Communications · OSTI ID:1909043

Lu, Shuo; Hu, Liya; Lin, Hanfeng; +7 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
antibiotics
combinatorial libraries
enzyme mechanisms
hydrolases
x-ray crystallography

Title: Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

Citation Formats

References (60)

Similar Records

Related Subjects