Mechanistic Studies of the Biosynthesis of 3,6‐Dideoxysugars in Bacteria: Exploration of a Novel C‐O Bond Cleavage Event

Abstract

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 E 1 andEj. We have now purified the wild-type enzymes, cloned the corresponding genes (ascC for.B, and ascDfor~), and overexpressed the gene products in Escherichia coli. The purified E] is aflavoprotein comprising an iron-sulfur Center and E] 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 I and E 3 must proceed via a radical mechanism. Recently, EPR analysis of El~catalysis indicated a potential new redox role for pyridoxamine as a cofactor. 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 which coenzyme B 6 may facilitate one-electron redox chemistry.Thus, the unprecedented mechanisms of E 1 and E 3 distinguish this system asa novel radical deoxygenation with potentially interesting future developments. 627

Protein Radicals in Enzyme Catalysis

“…383,385 The spectrum, however, is inconsistent with that of 13 and can be accommodated only by a hydroxylated, one-electron oxidized histidine radical (14). 386 How such a species would be generated was not addressed.…”

“…224 The Knappe mechanism has been strongly influenced by observations made with the formate analogue, hypophosphite, in the presence of Ac-CoA 16,198,212,214 or, in the reverse direction with the corresponding pyruvate analogue, acetyl phosphinate (Scheme 12). 213,225 Incubation of E a with [ 3 H]-H 3 PO 2 in the presence of [ 14 C]acetylCoA results in rapid inactivation, formation of volatile 3 H, presumably 3 H 2 O, and 14 C-labeled protein. The label is stable at neutral and acidic pH, and peptide mapping with chymotrypsin identified a 14 C-labeled peptide.…”

“…Studies of Boussac et al indicate that the amino acid radical gives rise to a visible spectrum with a λ max at about 315 nm, which they suggested to be associated with an oxidized histidine radical. 383,385 The spectrum, however, is inconsistent with that of 13 and can be accommodated only by a hydroxylated, one-electron oxidized histidine radical (14). 386 How such a species would be generated was not addressed.…”

“…We will limit the scope of this review to enzymatic systems that utilize amino acid or modified amino acid based radicals that are covalently linked to the protein. Other enzymatic systems in which the involvement of non-amino acid based organic radicals have been proposed such as the cytochrome P450 enzyme family, flavin and pyrroloquinoline quinone (PQQ) dependent enzymes, , heme peroxidases, the pyridoxal phosphate dependent 2,3-lysine aminomutase, , pyruvate:ferredoxin oxidoreductase, and enzymes involved in 3,6-dideoxyhexose biosynthesis have been extensively reviewed elsewhere. It is hoped that the general principles and recent experimental methods described herein will serve as a guide for examining new protein radical containing systems that will undoubtedly be discovered.…”

Identification of an Unusual [2Fe-2S]-Binding Motif in the CDP-6-deoxy-d-glycero-l-threo-4-hexulose-3-dehydrase from Yersinia pseudotuberculosis: Implication for C-3 Deoxygenation in the Biosynthesis of 3,6-Dideoxyhexoses

“…The most intriguing reaction in the biosynthesis of CDP-3,6-dideoxyhexoses is the C-3 deoxygenation step, in which CDP-6-deoxy-L-threo-D-glycero-4-hexulose (1, also known as CDP-4-keto-6-deoxy-D-glucose) is converted to CDP-3,6-dideoxy-D-glycero-Dglycero-4-hexulose (2, also known as CDP-4-keto-3,6dideoxy-D-glucose) (3)(4)(5)(6). This C-O bond cleavage event is catalyzed by two enzymes: CDP-6-deoxy-L-threo-Dglycero-4-hexulose-3-dehydrase (E 1 ) 1 and its reductase (E 3 ) (Scheme 1) (7).…”