The emergence and spread of antibiotic resistant bacterial pathogens has become an alarming problem. The recently described IMP-1 metallo-beta-lactamase, confers resistance to virtually all beta-lactam antibiotics. The focus of this proposal is to use site-directed mutagenesis to define the determinants of catalysis and substrate specificity of the class B metallo-beta-lactamase, IMP-1. The system will be developed using the following specific aims: 1) randomization of the putative critical residues of IMP-1 to test their role in carbapenamase substrate binding and hydrolysis; 2) identification of the determinants of IMP-1 substrate specificity and 3) determining if both zinc-binding sites are required for IMP-1 activity. The construction of a randomized site-directed mutagenesis library followed by the selection and sequencing of functional clones will assess the role of the target residues in these aims. Function is defined by the ability of the enzyme to hydrolyze beta-lactam antibiotics. The mutagenesis strategy will help define what amino acid residues are necessary for binding different substrates and zinc (II) by identifying substitutions that affect the binding of these molecules. Kinetic studies will be used to characterize mutant enzymes with altered specificity, and metal- binding and biochemical assays will establish a correlation between zinc content and catalytic activity of IMP-1.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31AI010535-02
Application #
6372931
Study Section
Special Emphasis Panel (ZRG1-BIOL-1 (02))
Program Officer
Hernandez, Milton J
Project Start
2001-08-01
Project End
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
2
Fiscal Year
2001
Total Cost
$26,222
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
Materon, Isabel C; Queenan, Anne Marie; Koehler, Theresa M et al. (2003) Biochemical characterization of beta-lactamases Bla1 and Bla2 from Bacillus anthracis. Antimicrob Agents Chemother 47:2040-2
Majiduddin, Fahd K; Materon, Isabel C; Palzkill, Timothy G (2002) Molecular analysis of beta-lactamase structure and function. Int J Med Microbiol 292:127-37
Materon, I C; Palzkill, T (2001) Identification of residues critical for metallo-beta-lactamase function by codon randomization and selection. Protein Sci 10:2556-65