MECHANISTIC STUDIES OF THE BIOSYNTHESIS OF 3,6-DIDEOXYSUGARS IN BACTERIA - EXPLORATION OF A NOVEL C-O BOND-CLEAVAGE EVENT

Deoxy sugars are ubiquitous in nature and contribute to diverse biolog ical activities. Attempts to design systems to control or to mimic the ir functions are hampered, however, by the lack of biosynthetic knowle dge of these unique sugars. To elucidate the mechanism by which the su gar deoxygenation is effected, we have initiated a study to explore th e biosynthesis of CDP-ascarylose, a 3,6-dideoxyhexose found in the lip opolysaccharides of Yersinia pseudotuberculosis, and our initial focus centered on C-3 deoxygenation catalyzed by E(1) and E(3). We have now purified the wild-type enzymes, cloned the corresponding genes (ascC for E(1) and ascD for E(3)), and overexpressed the gene products in Es cherichia coli. The purified E(3) is a flavoprotein comprising an iron -sulfur center and E(1) is an iron-sulfur containing, pyridoxamine 5'- phosphate-dependent enzyme. Since these iron-sulfur clusters are well known one-electron carriers, reactions mediated by E(1) and E(3) must proceed via a radical mechanism. Recently, EPR analysis of E(1)/E(3) c atalysis indicated a potential new redox role for pyridoxamine as a co factor. These findings make this system unique from two perspectives: E(1) is the only coenzyme B-6-dependent catalyst that interacts with a sugar and not with an amino acid, and it is the first example in whic h coenzyme B-6 may facilitate one-electron redox chemistry. Thus, the unprecedented mechanisms of E(1) and E(3) distinguish this system as a novel radical deoxygenation with potentially interesting future devel opments.