Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA

KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan

Nobuo Sugiura, Yuichi Baba, Yoshirou Kawaguchi, Toru Iwatani, Kiyoshi Suzuki, Takahiro Kusakabe, Kiwamu Yamagishi, Koji Kimata, Yoshimitsu Kakuta, Hideto Watanabe

研究成果: ジャーナルへの寄稿記事

22 引用 (Scopus)

抄録

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1-4 and GlcNAc α1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

元の言語英語
ページ(範囲)1597-1606
ページ数10
ジャーナルJournal of Biological Chemistry
285
発行部数3
DOI
出版物ステータス出版済み - 1 15 2010

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Glucuronosyltransferase
Heparitin Sulfate
Transferases
Escherichia coli
Association reactions
Enzymes
Bioengineering
Disaccharides
Multigene Family
Glycosaminoglycans
Aspartic Acid
Polymerization
Lysine
Extracellular Matrix
Polysaccharides
Glutamic Acid
Proteins
Biosynthesis
Enzyme activity
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA : KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan. / Sugiura, Nobuo; Baba, Yuichi; Kawaguchi, Yoshirou; Iwatani, Toru; Suzuki, Kiyoshi; Kusakabe, Takahiro; Yamagishi, Kiwamu; Kimata, Koji; Kakuta, Yoshimitsu; Watanabe, Hideto.

:: Journal of Biological Chemistry, 巻 285, 番号 3, 15.01.2010, p. 1597-1606.

研究成果: ジャーナルへの寄稿記事

Sugiura, Nobuo ; Baba, Yuichi ; Kawaguchi, Yoshirou ; Iwatani, Toru ; Suzuki, Kiyoshi ; Kusakabe, Takahiro ; Yamagishi, Kiwamu ; Kimata, Koji ; Kakuta, Yoshimitsu ; Watanabe, Hideto. / Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA : KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan. :: Journal of Biological Chemistry. 2010 ; 巻 285, 番号 3. pp. 1597-1606.
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title = "Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA: KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan",
abstract = "Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1-4 and GlcNAc α1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.",
author = "Nobuo Sugiura and Yuichi Baba and Yoshirou Kawaguchi and Toru Iwatani and Kiyoshi Suzuki and Takahiro Kusakabe and Kiwamu Yamagishi and Koji Kimata and Yoshimitsu Kakuta and Hideto Watanabe",
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T1 - Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA

T2 - KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan

AU - Sugiura, Nobuo

AU - Baba, Yuichi

AU - Kawaguchi, Yoshirou

AU - Iwatani, Toru

AU - Suzuki, Kiyoshi

AU - Kusakabe, Takahiro

AU - Yamagishi, Kiwamu

AU - Kimata, Koji

AU - Kakuta, Yoshimitsu

AU - Watanabe, Hideto

PY - 2010/1/15

Y1 - 2010/1/15

N2 - Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1-4 and GlcNAc α1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

AB - Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1-4 and GlcNAc α1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

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