# BETA LACTAMASE MUTATIONS IN ANTIBIOTIC RESISTANCE

> **NIH NIH R01** · BAYLOR COLLEGE OF MEDICINE · 2020 · $396,250

## Abstract

Project Summary/Abstract
Bacterial resistance to antimicrobial agents has increased in recent years, posing a significant threat
to antibiotic therapy. β-lactam antibiotics are the most often prescribed antibacterial agents. The most
common mechanism of resistance is β-lactamase-catalyzed hydrolysis, which renders the antibiotics
ineffective. Because of the diverse range of substrate specificities of these enzymes, virtually all
β-lactam antibiotics are susceptible to hydrolysis. The design of new antibiotics that escape
hydrolysis by the growing collection of β-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 β-lactamases. The CTX-M β-lactamases comprise a
family of extended-spectrum β-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 β-lactam antibiotics. Finally, KPC-2 β-lactamase is able to hydrolyze nearly all
β-lactam antibiotics including carbapenems. It is not known, however, how KPC-2 is able to
hydrolyze carbapenems while other class A β-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.

## Key facts

- **NIH application ID:** 9837405
- **Project number:** 5R01AI032956-28
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Timothy Palzkill
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $396,250
- **Award type:** 5
- **Project period:** 1992-07-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9837405

## Citation

> US National Institutes of Health, RePORTER application 9837405, BETA LACTAMASE MUTATIONS IN ANTIBIOTIC RESISTANCE (5R01AI032956-28). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9837405. Licensed CC0.

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