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The Journal of biological chemistry

A bifunctional enzyme with L-fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-fucose in Arabidopsis.


PMID 18199744

Abstract

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 Gly(133) or Gly(830) 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.

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