D-ARABINOSE SYNTHESIS AS A TARGET SITE FOR CHEMOTHERAPY

点击翻译按钮获取中文摘要

DESCRIPTION (adapted from application abstract): D-Arabinose is a major sugar of the mycobacterial cell wall and occurs in two important cell wall polymers, an arabinogalactan and a lipoarabinomannan. In spite of the widespread occurrence of this sugar in this group of organisms, very little is known about its pathway of biosynthesis. Previous studies from our laboratory and that of McNeil et al, in which mycobacterial cells were incubated with [1-(14)C]- or - [6-(14)C] glucose indicated that both labels were equally good precursors of radioactive arabinose, suggesting that this sugar arises via the degradation and reassembly of a hexose precursor, rather than directly by the hexose-monophosphate shunt. However, the specific steps leading to an arabinose-phosphate or an activated form of D-arabinose are unknown, or not well documented. Thus, while the Brennan group have definitely identified an undecaprenyl-P-arabinose as the immediate precuror of arabinose polymers, steps prior to formation of the lipid are unclear. Published reports on the nature of the nucleoside diphosphate arabinose are conflicting. Since D-arabinose does not occur in any animal cells, any of the steps in its synthesis or activation represent excellent target sites for chemotherapy. Thus, in this pilot project, we will explore the reactions involved in synthesis and activation of D-arabinose. First, we will isolate and characterize the nucleoside diphosphate D-arabinose from whole cells of mycobacteria, in order to determine the nature of the nucleotide base (one report in the literature indicates it is guanosine, another that it is uridine). In these cell extracts, we will also look for the presence of arabinose-5-P and arabinose-1-P to verify the pathway of synthesis. Then, we will incubate cell free extracts of mycobacteria with radioactive arabinose-5-P and arabinose-1-P to assay for the presence of a phosphoarabinose mutase and an XDP-arabinose pyrophosphorylase. These enzymes will be purified and characterized and tested with various potential inhibitors that may be useful chemotherapeutic agents.