Y. Yoshida, Y. Nakano, T. Nezu
Jun 11, 1999
Citations
5
Influential Citations
45
Citations
Journal
The Journal of Biological Chemistry
Abstract
The serotype-specific polysaccharide antigen ofActinobacillus 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 byd-glucose-1-phosphate thymidylyltransferase and dTDP-d-glucose 4,6-dehydratase, which are encoded byrmlA 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 theK 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.