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The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.