UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.
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Research Article|
November 11 2009
Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants Available to Purchase
Toshihisa Kotake;
Toshihisa Kotake
1
*Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
†Institute for Environmental Science and Technology, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
1To whom correspondence should be addressed ([email protected]).
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Ryohei Takata;
Ryohei Takata
*Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Rajeev Verma;
Rajeev Verma
‡Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, U.S.A.
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Masato Takaba;
Masato Takaba
*Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Daisuke Yamaguchi;
Daisuke Yamaguchi
§Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Takahiro Orita;
Takahiro Orita
*Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Satoshi Kaneko;
Satoshi Kaneko
∥Food Biotechnology Division, National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
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Koji Matsuoka;
Koji Matsuoka
¶Division of Material Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Tetsuo Koyama;
Tetsuo Koyama
¶Division of Material Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Wolf-Dieter Reiter;
Wolf-Dieter Reiter
‡Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, U.S.A.
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Yoichi Tsumuraya
Yoichi Tsumuraya
*Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
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Publisher: Portland Press Ltd
Received:
July 06 2009
Revision Received:
September 03 2009
Accepted:
September 15 2009
Accepted Manuscript online:
September 15 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 424 (2): 169–177.
Article history
Received:
July 06 2009
Revision Received:
September 03 2009
Accepted:
September 15 2009
Accepted Manuscript online:
September 15 2009
Citation
Toshihisa Kotake, Ryohei Takata, Rajeev Verma, Masato Takaba, Daisuke Yamaguchi, Takahiro Orita, Satoshi Kaneko, Koji Matsuoka, Tetsuo Koyama, Wolf-Dieter Reiter, Yoichi Tsumuraya; Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants. Biochem J 1 December 2009; 424 (2): 169–177. doi: https://doi.org/10.1042/BJ20091025
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