A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose.
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 Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, 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.
Yoshida
Y
Y
Department of Preventive Dentistry, Kyushu University Faculty of Dentistry, Fukuoka 812-8582, Japan. yosh@dent.kyushu-u.ac.jp
Nakano
Y
Y
Nezu
T
T
Yamashita
Y
Y
Koga
T
T
D016976:Aggregatibacter actinomycetemcomitans / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification; D005810:Multigene Family; D019906:Nuclear Magnetic Resonance, Biomolecular; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis; D015183:Restriction Mapping; D012703:Serotyping; D013237:Stereoisomerism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000096:biosynthesis*; D001426:Bacterial Proteins
D002237:Carbohydrate Dehydrogenases; D002238:Carbohydrate Epimerases; D006836:Hydro-Lyases; D009702:Nucleoside Diphosphate Sugars; D009713:Nucleotidyltransferases; D011135:Polysaccharides, Bacterial; D013401:Sugar Alcohol Dehydrogenases; D013942:Thymine Nucleotides; C119709:thymidine diphosphate fucose; C119708:thymidine diphosphate-4-keto-6-deoxy-D-glucose reductase; C107144:dTDP-4-dehydrorhamnose reductase; C020587:glucose-1-phosphate thymidylyltransferase; C022771:dTDPglucose 4,6-dehydratase; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D001426:Bacterial Proteins