Bacterial resistance to antimicrobial agents has increased in recent years, posing a significant threat to antibiotic therapy. ?-lactam antibiotics are the most often prescribed antibacterial agents. The most common mechanism of resistance is ?-lactamase-catalyzed hydrolysis, which renders the antibiotics ineffective. Because of the diverse range of substrate specificities of these enzymes, virtually all ?-lactam antibiotics are susceptible to hydrolysis. The design of new antibiotics that escape hydrolysis by the growing collection of ?-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of these enzymes. The objective of this application is to understand how amino acid sequence determines the structure, activity and evolution of class A ?-lactamases. The CTX-M ?-lactamases comprise a family of extended-spectrum ?-lactamases (ESBLs) that efficiently hydrolyze the oxyimino-cephalosporin cefotaxime but not ceftazidime. Certain amino acid substitutions in resistant clinical isolates, however, have been associated with increased ceftazidime hydrolysis. The goal of Aim 1 is to understand the structural and biochemical basis for the hydrolysis of ceftotaxime by CTX-M enzymes as well as the altered catalytic activity observed in CTX-M variants. In addition, studies from the current funding period indicate that cooperative interactions between amino acid residues in and near the active site play an important role in determining CTX-M substrate specificity. How active site residues cooperate to catalyze reactions is an important question with regard to how enzymes function. It is well-established that certain active site residues work together, cooperatively, to achieve catalysis. However, the extent to which the active site functions as a highly cooperative, interconnected unit versus the extent to which it is modular is unknown. The extent of cooperativity between substitutions will be addressed systematically in Aim 2 using random mutagenesis of active site residues of CTX-M-14 in combination with deep sequencing of mutants selected for the ability to hydrolyze various ?-lactam antibiotics. Finally, KPC-2 ?-lactamase is able to hydrolyze nearly all ?-lactam antibiotics including carbapenems. It is not known, however, how KPC-2 is able to hydrolyze carbapenems while other class A ?-lactamases, although similar in structure to KPC-2, cannot. An unbiased genetic approach will be used to identify mutants specifically defective in carbapenem hydrolysis. This approach has the advantage of surveying for changes anywhere in the enzyme that decrease carbapenem hydrolysis. Detailed biochemical and structural characterization of the mutant enzymes will address the mechanism behind the unique catalytic properties of class A carbapenemases.

Public Health Relevance

?-lactamases are enzymes that hydrolyze ?-lactam antibiotics to provide bacterial drug resistance and the emergence of ?-lactamases that are capable of hydrolyzing virtually all ?-lactam antibiotics is a significant threat to the efficacy of antibiotic therapy. The proposed experiments utilize genetic analysis, enzyme kinetics, X-ray crystallography and high throughput sequencing of combinatorial amino acid substitution libraries to study the structure and function of two clinically important ?-lactamases. The results will provide detailed knowledge of how amino acid residue positions contribute to ?-lactam hydrolysis, which will facilitate understanding the molecular basis of the evolution of antibiotic resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032956-27
Application #
9608697
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Huntley, Clayton C
Project Start
1992-07-01
Project End
2021-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
27
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Sun, Zhizeng; Hu, Liya; Sankaran, Banumathi et al. (2018) Differential active site requirements for NDM-1 ?-lactamase hydrolysis of carbapenem versus penicillin and cephalosporin antibiotics. Nat Commun 9:4524
Palzkill, Timothy (2018) Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC ?-lactamases. Front Mol Biosci 5:16
Patel, Meha P; Hu, Liya; Brown, Cameron A et al. (2018) Synergistic effects of functionally distinct substitutions in ?-lactamase variants shed light on the evolution of bacterial drug resistance. J Biol Chem 293:17971-17984
Adamski, Carolyn J; Palzkill, Timothy (2017) Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A ?-lactamases. BMC Biochem 18:2
Patel, Meha P; Hu, Liya; Stojanoski, Vlatko et al. (2017) The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M ?-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation. Biochemistry 56:3443-3453
Adamski, Carolyn J; Palzkill, Timothy (2017) BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A ?-Lactamases. Biochemistry 56:1075-1084
Stojanoski, Vlatko; Adamski, Carolyn J; Hu, Liya et al. (2016) Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine ?-Lactamases by Relieving Steric Strain. Biochemistry 55:2479-90
Chow, Dar-Chone; Rice, Kacie; Huang, Wanzhi et al. (2016) Engineering Specificity from Broad to Narrow: Design of a ?-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC ?-Lactamase. ACS Infect Dis 2:969-979
Adamski, Carolyn J; Cardenas, Ana Maria; Brown, Nicholas G et al. (2015) Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ?-lactamases. Biochemistry 54:447-57
Patel, Meha P; Fryszczyn, Bartlomiej G; Palzkill, Timothy (2015) Characterization of the global stabilizing substitution A77V and its role in the evolution of CTX-M ?-lactamases. Antimicrob Agents Chemother 59:6741-8

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