The goal of the proposed research is to characterize at the molecular level how mutations can alter the structure and substrate specificity of the clinically important TEM-1 beta-lactamase and also to develop new inhibitors of the enzymes. Specific objectives include: 1. Identify the amino acid residues that control beta-lactamase substrate specificity. Every amino acid position in the TEM-1 beta-lactamase has been randomized to sample all possible amino acid substitutions. All of the random substitutions will be screened to identify those amino acid substitutions that increase the catalytic activity of the enzyme for a number of beta-lactam antibiotics. In addition, mosaic enzymes between TEM-1 and the class A Mycobacterium fortuitum beta-lactamase will be constructed to identify the residues that are responsible for the large difference in substrate profile between these enzymes. 2. Determine the mechanism by which amino acid substitutions alter beta-lactamase structure and substrate specificity. Biochemical methods will be used to further characterize the catalytic properties of the specificity mutants. These studies will include purification of the mutant enzymes and determination of kinetic parameters. In addition, the mechanism of action of a set of substitutions in an active-site loop that were previously shown to alter beta- lactamase substrate specificity will be investigated by genetic and biochemical approaches. 3. Use combinatorial library technology to develop new inhibitors of beta-lactamase. Random peptide phage display technology will be used to develop new TEM-1 beta- lactamase inhibitors. In addition to providing an important research tool, these inhibitors may be useful in antibiotic therapy or may serve as lead compounds for the design of new inhibitors of beta-lactamase. In total, these studies should contribute to the long term goal of understanding how beta-lactamase will respond to the selective pressure of antibiotic therapy with the hope that such knowledge will lead to the design of new drugs and inhibitors that are immune to this response.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032956-09
Application #
6170155
Study Section
Special Emphasis Panel (ZRG5-BM-1 (04))
Project Start
1992-07-01
Project End
2001-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
9
Fiscal Year
2000
Total Cost
$238,398
Indirect Cost
Name
Baylor College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
074615394
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
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
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
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

Showing the most recent 10 out of 71 publications