| Peptidoglycan is a cross-linked polymer matrix surrounding bacterial cells to provide rigidity and strength for the cell. Peptidoglycan is essential for the survival of bacteria and many antibiotics function by interfering its biosynthesis. Due to recent surges of antibiotic resistance, it becomes imperative to understand more about the enzymes involved in peptidoglycan biosynthesis. Unfortunately, many of these enzymes are difficult to study because their substrates are either not available or not soluble under suitable assay conditions. MurG, a cytoplasmic membrane-associated enzyme, catalyzes the transfer of an N-acetyl glucosamine sugar from an UDP-GlcNAc donor to its undecaprenyl-pyrophosphoryl-N-acetylmuramyl-pentapeptide substrate, known as Lipid I, to form Lipid II, which is the building block of peptidoglycan. Although MurG is essential for peptidoglycan biosynthesis and a potential target for novel antibiotic design, there was no structural or mechanistic information about the enzyme because of the difficulties in isolating its substrate, Lipid I, from natural sources. Therefore, we turned to synthetic chemistry for Lipid I analogues.; We developed a synthetic route of a Lipid I analogue, in which a 10-carbon lipid chain with a α-saturated isoprenyl unit was used to replace the 55-carbon lipid chain in natural Lipid I which may add difficulties to both the synthesis and biological assays. The target Lipid I analogue was proven active for MurG. This achievement has enabled the purification of MurG and the development of an aqueous enzyme assay. The synthetic route was also proven convergent and allowed us to synthesize a variety of Lipid I analogues. These analogues were utilized in characterizing the kinetic properties of the enzyme.; There was little information available about the enzymes involved in later stage of peptidoglycan biosynthesis because of the difficulties in obtaining their substrate, Lipid II. Using purified MurG and its substrate analogue, a chemo-enzymatic synthesis of Lipid II was developed. With the synthetic Lipid II analogue, we found direct evidence that ramoplanin, an antibiotic proposed to function by interaction with Lipid I, binds with higher affinity to Lipid II. It allowed us to proposed a new mechanism for ramoplanin. This synthetic route was also proved effective in synthesizing a variety of Lipid II analogues which could be utilized in studying transglycosylases, the enzymes that polymerize the disaccharide of Lipid II. |
