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J. Biol. Chem., Vol. 283, Issue 13, 8125-8135, March 28, 2008

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A Bifunctional Enzyme with L-Fucokinase and GDP-L-fucose Pyrophosphorylase Activities Salvages Free L-Fucose in Arabidopsis^*

Toshihisa Kotake¹, Sachiko Hojo, Noriaki Tajima, Koji Matsuoka^¶, Tetsuo Koyama^¶, and Yoichi Tsumuraya

From the Division of Life Science, Graduate School of Science and Engineering, and Department of Biochemistry and Molecular Biology, Faculty of Science, ^¶Division of Material Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan

Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because L-fucose (L-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for L-Fuc are expected to be independent from those for Glc, Gal, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene (designated AtFKGP) of a bifunctional enzyme with similarity to both L-fucokinase and GDP-L-Fuc pyrophosphorylase. Recombinant AtFKGP (rAt-FKGP) expressed in Escherichia coli showed both L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, generating GDP-L-Fuc from L-Fuc, ATP, and GTP as the starting substrates. Point mutations in rAtFKGPs at either Gly¹³³ or Gly⁸³⁰ caused loss of GDP-L-Fuc pyrophosphorylase and L-fucokinase activity, respectively. The apparent K_m values of L-fucokinase activity of rAtFKGP for L-Fuc and ATP were 1.0 and 0.45 mM, respectively, and those of GDP-L-Fuc pyrophosphorylase activity for L-Fuc 1-phosphate and GTP were 0.052 and 0.17 mM, respectively. The expression of AtFKGP was detected in most cell types of Arabidopsis, indicating that salvage reactions for free L-Fuc catalyzed by AtFKGP occur ubiquitously in Arabidopsis. Loss-of-function mutants with tDNA insertion in AtFKGP exhibited higher accumulation of free L-Fuc in the soluble fraction than the wild-type plant. These results indicate that AtFKGP is a bifunctional enzyme with L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, which salvages free L-Fuc in Arabidopsis.

Received for publication, December 11, 2007

^* This work was supported in part by a grant for ground research for space utilization (to T. K.) from the Japan Space Forum and a Grant-in-aid for Scientific Research 17770028 (to T. K.) from Ministry of Education, Culture, Sports, Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed. Fax: 81-48-858-3384; E-mail: kotake{at}molbiol.saitama-u.ac.jp.

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