Objective
THE BACTERIAL TARGETS OF THE B-LACTAM ANTIBIOTICS (PENICILLINS AND CEPHALOSPORINS) ARE MEMBRANE-BOUND DD-PEPTIDASES. HOWEVER, THE RAPID SPREAD IN THE BACTERIAL WORLD OF GENES CODING FOR VARIOUS TYPES OF B-LACTAM HYDROLYSING B-LACTAMASES QUICKLY "OUTDATES" A PARTICULAR ANTIBIOTIC. USING THE INFORMATION OBTAINED FROM THE UNRAVELING OF STRUCTURE-ACTIVITY RELATIONSHIPS OF THESE TWO TYPES OF ENZYMES IT IS EXPECTED TO DESIGN NOVEL TYPES OF INACTIVATORS.
Active-site serine DD-peptidases/penicillin-binding proteins (PBP) are enzymes essential to the survival of bacteria. Their inactivation by penicillins results in bacterial death. Active site serine beta-lactamases are also produced by bacteria; they destroy penicillins. The 2 types of enzymes share structural and catalytic characteristics.
Research was carried out in order to understand the mechanisms of action of these 2 types of enzymes, with the hope of being able to design antibiotic molecules which might inactivate the DD-peptidases and PBPs,while escaping destruction by the beta-lactamases.
The primary approaches involved:
the determination of the 3-dimensional structures of various DD-peptidases and PBPs and beta-lactamases;
the study of their enzymatic properties and those of mutants obtained by site directed mutagenesis;
the molecular modelling of the catalyzed reaction pathways.
Based on the resolved 3-dimensional structures, superimposition of the Streptomyces R61 DD-peptidases and PBP over Streptomyces albus G beta-lactamase shows striking similarities at the level of the active sites. The tetrade serine*62-valine-threonine-lysine 65 (serine*48-valine-phenylalanine-lysine 51 in the beta-lactamase; where serine* = active site serine) that forms part of the alpha-helix H2, and the triad histidine 298-threonine-glycine 300 (lysine 218-threonine-glycine 220 in the beta-lactamase) that occurs on the innermost strand S3 of the beta-sheet, are almost superimposable. A conserved loop tryosine 159-serine-asparagine 161 (serine 114-aspartic acid-asparagine 116 in the beta-lactamase) forms the other side of the enzyme active sites, and a negatively charged residue aspartic acid 225 (glutamic acid 150 in the beta-lactamase) occurs in equivalent positions. However, the loop in the beta-lactamase and the aspartic acid in the DD-peptidase are located closer to the active site serine. The role of these residues in terms of catalysis and protein stability has been investigated by site directed mutagenesis.
TO UNRAVEL THE STRUCTURE-ACTIVITY RELATIONSHIPS OF THE PENICILLIN-SENSITIVE DD-PEPTIDASE FROM STREPTOMYCES R61 AND THE CLASS A
B-LACTAMASE FROM BACILUS LICHENIFORMIS AND OTHER BACTERIA. THE RESEARCH INVOLVES :
- REFINEMENT OF THE 3D-STRUCTURE;
- ANALYSIS OF THE ROLE OF SPECIFIC AMINO ACID RESIDUES IN THE BINDING OF SUBSTRATES;
- INVESTIGATION OF THE CATALYTIC MECHANISM;
- MUTAGENESIS OF IMPORTANT AMINO-ACID RESIDUES;
- STUDY OF THE CATALYTIC AND STRUCTURAL EFFECTS INDUCED BY THESE MODIFICATIONS.
THE WORK SHOULD LEAD TO MOLECULAR MODELLING OF BOTH THE CATALYSED REACTION PATHWAY AND THE ENZYMES'ACTIVES SITES.
Fields of science
- natural sciencesbiological sciencesmicrobiologybacteriology
- medical and health sciencesbasic medicinepharmacology and pharmacypharmaceutical drugsantibiotics
- natural scienceschemical sciencescatalysis
- natural scienceschemical sciencesorganic chemistryamines
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
Programme(s)
Topic(s)
Data not availableCall for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
4000 LIEGE
Belgium