This application is concerned with the crystallographic aspects of a collaborative effort to study the origin of bacterial resistance to beta- lactam antibiotics related to beta-lactamase. The other parts of the collaboration include the molecular biology of beta-lactamase, in particular site directed mutagenesis, the biochemistry aspects, and the molecular modeling of the system. The study is relevant to the recovery of the effectiveness of penicillin and cephalosporin therapy, which has been rapidly diminishing due to the ability of pathogenic bacteria to produce beta-lactamases. These enzymes hydrolyze the beta-lactam ring prior to the delivery of the drug to its target - the bacterial cell wall - where the penicillin sensitive enzymes responsible for the cell wall synthesis and repair are located. The understanding of the catalytic process and the substrate specificity at the atomic level is required for the development of new effective antibiotics. X-ray crystallography provides such accurate information. The high resolution crystal structure of beta-lactamase from Staphylococcus aureus PC1 enables us to carry out binding studies to various substrates and inhibitors. Cryocrystallography, aiming at trapping reaction intermediates at liquid nitrogen temperature, will be employed, as well as conventional room temperature measurements. Site directed mutants have been designed to test the proposed catalytic mechanism, alter substrate specificity, and probe the folding properties of the enzyme. These will be prepared for biochemical and crystallographic analysis. To address the problem of the recently acquired resistance to the potent """"""""third generation"""""""" cephalosporins, the crystal structures of the E. coli SHV1 and SHV2 beta-lactamases will be determined, the latter being responsible for the new resistance. The various aspects of the project will provide information pertaining not just to the specific clinical problem caused by beta-lactamases, but will also contribute to our fundamental understanding of protein function and stability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI027175-05
Application #
3141314
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1989-01-01
Project End
1997-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
Organized Research Units
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21202
Chen, C C; Herzberg, O (2001) Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine. Biochemistry 40:2351-8
Chen, C C; Herzberg, O (1999) Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp. Protein Eng 12:573-9
Banerjee, S; Pieper, U; Kapadia, G et al. (1998) Role of the omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase. Biochemistry 37:3286-96
Pieper, U; Kapadia, G; Mevarech, M et al. (1998) Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii. Structure 6:75-88
Pieper, U; Hayakawa, K; Li, Z et al. (1997) Circularly permuted beta-lactamase from Staphylococcus aureus PC1. Biochemistry 36:8767-74
Banerjee, S; Shigematsu, N; Pannell, L K et al. (1997) Probing the non-proline cis peptide bond in beta-lactamase from Staphylococcus aureus PC1 by the replacement Asn136 --> Ala. Biochemistry 36:10857-66
Chen, C C; Smith, T J; Kapadia, G et al. (1996) Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1. Biochemistry 35:12251-8
Zawadzke, L E; Chen, C C; Banerjee, S et al. (1996) Elimination of the hydrolytic water molecule in a class A beta-lactamase mutant: crystal structure and kinetics. Biochemistry 35:16475-82
Liao, D I; Kapadia, G; Ahmed, H et al. (1994) Structure of S-lectin, a developmentally regulated vertebrate beta-galactoside-binding protein. Proc Natl Acad Sci U S A 91:1428-32
Chen, C C; Rahil, J; Pratt, R F et al. (1993) Structure of a phosphonate-inhibited beta-lactamase. An analog of the tetrahedral transition state/intermediate of beta-lactam hydrolysis. J Mol Biol 234:165-78

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