A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose

Yasuo Yoshida, Yoshio Nakano, Takashi Nezu, Yoshihisa Yamashita, Toshihiko Koga

Research output: Contribution to journalArticle

34 Citations (Scopus)

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-D-fucose 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 reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas- liquid 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-D-glucose and NADPH are 97.3 and 28.7 μM, 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.

Original languageEnglish
Pages (from-to)16933-16939
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number24
DOIs
Publication statusPublished - Jun 11 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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