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

33 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

Fingerprint

Fucose
Oxidoreductases
Genes
Polysaccharides
Aggregatibacter actinomycetemcomitans
Enzymes
glucose-1-phosphate thymidylyltransferase
Multigene Family
Hydro-Lyases
Glucose
Rhamnose
Reverse-Phase Chromatography
Gene encoding
thymidine diphosphate fucose
Diphosphates
NADP
Liquid chromatography
High performance liquid chromatography
Gas Chromatography
Sugars

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose. / Yoshida, Yasuo; Nakano, Yoshio; Nezu, Takashi; Yamashita, Yoshihisa; Koga, Toshihiko.

In: Journal of Biological Chemistry, Vol. 274, No. 24, 11.06.1999, p. 16933-16939.

Research output: Contribution to journalArticle

Yoshida, Yasuo ; Nakano, Yoshio ; Nezu, Takashi ; Yamashita, Yoshihisa ; Koga, Toshihiko. / A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 24. pp. 16933-16939.
@article{f9687b7acc36409bb442a54dbcd01607,
title = "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-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.",
author = "Yasuo Yoshida and Yoshio Nakano and Takashi Nezu and Yoshihisa Yamashita and Toshihiko Koga",
year = "1999",
month = "6",
day = "11",
doi = "10.1074/jbc.274.24.16933",
language = "English",
volume = "274",
pages = "16933--16939",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "24",

}

TY - JOUR

T1 - A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose

AU - Yoshida, Yasuo

AU - Nakano, Yoshio

AU - Nezu, Takashi

AU - Yamashita, Yoshihisa

AU - Koga, Toshihiko

PY - 1999/6/11

Y1 - 1999/6/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0033546181&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033546181&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.24.16933

DO - 10.1074/jbc.274.24.16933

M3 - Article

VL - 274

SP - 16933

EP - 16939

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 24

ER -