| Bacterial cell wall biosynthesis is a complex and dynamic process involving participation of a variety of enzymes. It is an attractive target for development of antibiotics. However, the emergence of bacterial resistance to most antibiotics, including vancomycin, now represents a threat to public health. This rapid increase to resistance has led to a resurgence of interest in the study of the essential bacterial enzymes involved in peptidoglycan synthesis. An efficient chemo-enzymatic strategy has been developed for the synthesis of bacterial cell-wall precursor UDP-N-acetylmuramyl-peptides, including the vancomycin-resistant precursor UDP-N-acetylmuramyl-depsipentapeptide, and used in the development of a sensitive assay for identification of new antibiotics against vancomycin-resistant strains. This convergent strategy, starting with UDP-muramic acid prepared enzymatically and then coupled with a chemically synthesized pentapeptide, allows for preparation of various UDP- N-acetylmuramyl-peptides found in antibiotic-resistant bacteria.; MurG, a transglycosylase involved in the second stage of bacterial cell wall biosynthesis, catalyzes the transfer of N-acetylglucosamine to Lipid I forming Lipid II. A continuous fluorescence coupled enzymatic assay was developed to study the MurG acceptor specificity toward different Lipid I analogues with various substituents replacing the undecaprenyl moiety. Most of the Lipid I analogues were accepted as substrates and among these the saturated C14 analogue exhibited the best activity. This substrate was then used to evaluate the inhibition activity of such antibiotics as moenomycin, vancomycin, and two chlorobiphenyl vancomycin derivatives. A vancomycin derivative with the chlorobiphenyl moiety on the aglycon was identified as a potent inhibitor of MurG.; In stage III of bacterial cell wall biosynthesis, Lipid II is polymerized by the transglycosylase to form the glycan part of the peptidoglycan. Because differences between the glycan motif of wild-type and resistant strains have not been observed so far and the transglycosylase is located at the extracellular surface of the membrane, this polymerization reaction catalyzed by transglycosylase represents the most promising target to develop new antibiotics. A coupled fluorescence assay and a surface plasmon resonance (SPR) binding assay have been developed to evaluate potential inhibitors of this enzyme. |
