A bifunctional enzyme with L-fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-fucose in Arabidopsis.
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.
Kotake
Toshihisa
T
Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama, Japan. kotake@molbiol.saitama-u.ac.jp
Hojo
Sachiko
S
Tajima
Noriaki
N
Matsuoka
Koji
K
Koyama
Tetsuo
T
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation; D005643:Fucose / Q000378:metabolism*; D018745:Genome, Plant / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017354:Point Mutation / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D005643:Fucose; D017853:Phosphotransferases (Alcohol Group Acceptor); C020153:fucokinase; C115708:GDP-L-fucose pyrophosphorylase; D009713:Nucleotidyltransferases