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 biological activities. Attempts to design systems to control or to mimic their functions are hampered, however, by the lack of biosynthetic knowledge of these unique sugars. To elucidate the mechanism by which the sugar deoxygenation is effected, we have initiated a study to explore the biosynthesis of CDP‐ascarylose, a 3,6‐dideoxyhexose found in the lipopolysaccharides of Yersinia pseudotuberculosis, and our initial focus centered on C‐3 deoxygenation catalyzed by E₁ and E₃. We have now purified the wild‐type enzymes, cloned the corresponding genes (ascC for E₁ and ascD for E₃), and overexpressed the gene products in Escherichia coli. The purified E₃ is a flavoprotein comprising an iron‐sulfur center and E₁ is an iron‐sulfur containing, pyridoxamine 5′‐phosphate‐de‐pendent enzyme. Since these iron‐sulfur clusters are well known one‐electron carriers, reactions mediated by E₁ and E₃ must proceed via a radical mechanism. Recently, EPR analysis of E₁/E₃ catalysis indicated a potential new redox role for pyridoxamine as a cofactor. These findings make this system unique from two perspectives: E₁ is the only coenzyme B₆‐dependent catalyst that interacts with a sugar and not with an amino acid, and it is the first example in which coenzyme B₆ may facilitate one‐electron redox chemistry. Thus, the unprecedented mechanisms of E₁ and E₃ distinguish this system as a novel radical deoxygenation with potentially interesting future developments.