A Novel NDP-6-deoxyhexosyl-4-ulose Reductase in the Pathway for the Synthesis of Thymidine Diphosphate-d-fucose

Abstract

The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-Dfucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversedphase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gasliquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the K m values for dTDP-4-keto-6-deoxy-Dglucose and NADPH are 97.3 and 28.7 M, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.Capsular polysaccharides are the outermost structures on a bacterial cell and play a critical role in the interactions of the cell with the environment (1). These molecules are prominent, structurally and serologically diverse antigens that are involved in pathogenic processes and in mediating resistance to important host defense mechanisms. In the case of human pathogens, a large number of different capsule serotypes have been identified (1). Previously, we reported that the serotype b-specific antigen of Actinobacillus actinomycetemcomitans Y4 is a capsular polysaccharide-like antigen consisting of two deoxyhexoses, L-rhamnose and D-fucose (2).A. actinomycetemcomitans is a nonmotile, Gram-negative, capnophilic, fermentative coccobacillus that has been implicated in the etiology and pathogenesis of localized juvenile periodontitis (3-5), adult periodontitis (6), and severe nonoral human infections (7). A. actinomycetemcomitans strains isolated from the human oral cavity are divided into five serotypes, a, b, c, d, and e (8 -10). The serologic specificity is defined by the polysaccharide on the surface of this organism (11), and the serotype-specific antigen is one of the immunodominant antigens in the organism (12, 13).In general, L-rhamnose is a component of many bacterial polysaccharides (14). The synthesis of thymidine diphosphate (dTDP) 1 -L-rhamnose, which is the activated nuc...

FlaA1, a New Bifunctional UDP-GlcNAc C6Dehydratase/ C4 Reductase from Helicobacter pylori

“…Since 4-keto intermediates are unstable molecules (34,47), their existence has only been demonstrated directly in a few instances (33,36). In the case of FlaA1, taking advantage of the high resolution and analytical power of CE and MS, we were able to provide direct evidence for the existence of a 4-keto, 6-methyl intermediate and follow the kinetics of its appearance.…”

Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans

“…4A) (19). These genes included rmlA, encoding glucose-1-phosphate thymidylyl transferase, which catalyzes the first step in the biosynthesis of dTDP-L-rhamnose, the activated nucleotide sugar precursor of A. actinomycetemcomitans O-PS carbohydrates (24,36); wzt, encoding the hydrophilic ATPase component of an ATP-binding cassette (ABC) membrane transporter involved in the export and assembly of lipopolysaccharide components (5); and dspA, encoding a putative glycosyltransferase (19). All three biofilm dispersal mutants exhibited wild-type surface adherence properties in broth (Fig.…”

N-Acetylmuramic Acid as Capping Element of α-D-Fucose-containing S-layer Glycoprotein Glycans from Geobacillus tepidamans GS5–97T

“…L-Rhamnose is a frequent constituent of bacterial polysaccharides, including Slayer glycans (2,25). D-Fucose, on the other hand, is a rare constituent of bacterial polysaccharides, which is almost exclusively present in LPS O-antigens (26,27). Because in S-layer glycans of Gram-positive bacteria typical sugar constituents of LPS are frequently found, it is reasonable to assume that in Gram-positive bacteria the ability to glycosylate proteins was acquired during evolution by lateral gene transfer from Gramnegative organisms.…”