A truly multidisciplinary approach to the study of the TEM-1 beta- lactamase, the prototypic class A beta-lactamase, has been outlined. The proposed research benefits from the expertise of the grant applicants in protein chemistry, mechanistic enzymology, computer-assisted molecular modeling, enzyme kinetics, microbiology, and molecular biology. The analysis of the active site crystal structure for the structurally-similar Bacillus licheniformis enzyme, in conjunction with kinetics of rationally- designed point mutant proteins at position 244 was instrumental in the discovery of a completely new set of interactions between the substrate(s) and the enzyme active site. Based on these analyses a new mechanism for active site anchoring of substrates involving hydrogen bonding by the side chains of Ser-130, Ser-235, and Arg-244 to the substrate carboxylate is presented. From kinetic experiments with purified enzymes, the contribution of the binding energy of each of these residues towards catalytic hydrolysis of both penicillins and cephalosporins will be evaluated. For these experiments traditional oligonucleotide-directed mutagenesis and the recently proposed in vitro substitution of unnatural amino acids will be performed. All proposed mutants have been designed with the assistance of computer graphics, giving full attention to interactions of the side chains to both substrate(s) and protein itself. It is expected that a complete set of energetics of interactions of substrates with residues Ser-130, Ser-235, and Arg-244 will emerge from these studies. A novel strategy for selection of mutant proteins with random amino acid substitutions exclusively at residues 130, 235, and 244 which, unlike other class A beta-lactamases, would turn over cephalosporins better than penicillins is described. This technique, presented here for the first time, will be a powerful tool in screening/selecting novel mutant enzymes with desired catalytic property(ies), and will have general applicability to other enzymes as well. Using molecular modeling we have identified the possibility of engineering a catalytic triad (similar to that of serine proteases) into the active site of the TEM-1 beta-lactamase. A discussion of the properties of this mutant enzyme is presented. We have reassessed the chemistry of beta-lactamase inactivation by the clinically useful mechanism-based inactivators, clavulanic acid and sulbactam. Our new structural insight suggests a central role for Arg-244 in the process of inactivation. Similarly, Arg-244 is proposed to play the role of a general acid in the tautomerization of delta2 -> delta1 pyrroline of carbapenem antibiotics. These proposals will be examined in both kinetic and computer-assisted graphics studies to delineate the details of the discrete chemical steps.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI033170-03
Application #
2068160
Study Section
Biochemistry Study Section (BIO)
Project Start
1992-08-01
Project End
1995-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Kumarasiri, Malika; Llarrull, Leticia I; Borbulevych, Oleg et al. (2012) An amino acid position at crossroads of evolution of protein function: antibiotic sensor domain of BlaR1 protein from Staphylococcus aureus versus clasS D ?-lactamases. J Biol Chem 287:8232-41
Borbulevych, Oleg; Kumarasiri, Malika; Wilson, Brian et al. (2011) Lysine Nzeta-decarboxylation switch and activation of the beta-lactam sensor domain of BlaR1 protein of methicillin-resistant Staphylococcus aureus. J Biol Chem 286:31466-72
Llarrull, Leticia I; Toth, Marta; Champion, Matthew M et al. (2011) Activation of BlaR1 protein of methicillin-resistant Staphylococcus aureus, its proteolytic processing, and recovery from induction of resistance. J Biol Chem 286:38148-58
Llarrull, Leticia I; Prorok, Mary; Mobashery, Shahriar (2010) Binding of the gene repressor BlaI to the bla operon in methicillin-resistant Staphylococcus aureus. Biochemistry 49:7975-7
Lemaire, Sandrine; Fuda, Cosimo; Van Bambeke, Francoise et al. (2008) Restoration of susceptibility of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics by acidic pH: role of penicillin-binding protein PBP 2a. J Biol Chem 283:12769-76
Totir, Monica A; Cha, Jooyoung; Ishiwata, Akihiro et al. (2008) Why clinically used tazobactam and sulbactam are poor inhibitors of OXA-10 beta-lactamase: Raman crystallographic evidence. Biochemistry 47:4094-101
Meroueh, Samy O; Bencze, Krisztina Z; Hesek, Dusan et al. (2006) Three-dimensional structure of the bacterial cell wall peptidoglycan. Proc Natl Acad Sci U S A 103:4404-9
Fisher, Jed F; Meroueh, Samy O; Mobashery, Shahriar (2005) Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity. Chem Rev 105:395-424
Thomas, Veena L; Golemi-Kotra, Dasantila; Kim, Choonkeun et al. (2005) Structural consequences of the inhibitor-resistant Ser130Gly substitution in TEM beta-lactamase. Biochemistry 44:9330-8
Golemi-Kotra, Dasantila; Meroueh, Samy O; Kim, Choonkeun et al. (2004) The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins. J Biol Chem 279:34665-73

Showing the most recent 10 out of 16 publications