Nucleotide sugar production in plants

Activated nucleotide sugars that serve as glycosyl donors for polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are generated through de novo pathway, in which various UDP- and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates. In the salvage pathway, an alternative route to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of respective nucleotide triphosphates as co-substrates (Figure 1). We isolated an enzyme with activity to convert various sugar 1-phosphate to respective UDP-sugar from pea sprouts. Recombinant USP expressed in Escherichia coli (Figure 2) can also form UDP-Glc, UDP-Gal, UDP-Xyl, UDP-L-Ara, UDP-GlcA,and UDP-GalA. In terestingly, USP does not act on Man 1-P and L-Fuc 1-P as they are metabolized as GDP-sugars. For the salvage reaction of free L-Fuc, L-fucokinase/GDP-L-Fuc pyrophosphorylase (FKGP) is working in plants. These enzymes are belonging to nucleotide sugar pyrophosphorylase family, and USP is distinct from known UDP-sugar making enzymes such as UDP-Glc pyrophosphorylase (Figure 3). References Kotake et al. (2016) J. Plant Res. 129: 781-792 (abstract) Sawake et al. (2015) Plant Cell 27: 3397-3409 (abstract) Kotake et al. (2008) J. Biol. Chem. 283: 8125-8135 (abstract) Kotake et al. (2007) Biosci. Biotechnol. Biochem. 71: 761-771 (abstract) Kotake et al. (2004) J. Biol. Chem. 279: 45728-45736 (abstract)

AGP degrading enzymes

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans found in all tissues of higher plants and localized in cell walls, plasma membranes, and the extracellular matrix. Although the functions of AGPs have not been clearly identified, several lines of evidence indicate that they are involved in many physiological events such as cell division, cell expansion, and cell death. AGPs are characterized by large amounts of carbohydrate components rich in galactose and L-arabinose, and protein components (core proteins: generally less than 10% of total weight) rich in hydroxyproline, serine, threonine, alanine, and glycine. The reducing galactosyl residues in the carbohydrate chains are attached through O-glycosyl linkages to Hyp and/or Ser/Thr residues. Since a large number of putative protein cores exist, it is difficult to separate particular AGP molecules from other AGP species in plant tissues, which make it difficult to elucidate the precise characteristics of individual AGPs. The carbohydrate moieties of AGPs have a common structure consisting of β-1,3-galactosyl backbones to which side chains of β-1,6-galactosyl residues are attached through O-6. L-Arabinose and lesser amounts of other auxiliary sugars such as glucuronic acid, 4-O-methyl-glucuronic acid, L-rhamnose, and L-fucose are attached to the side chains, usually at nonreducing terminals. It is important to study carbohydrate degrading enzymes of AGPs because hydrolytic enzymes specific to particular sugar residues and type of glycosidic linkage provide useful tools for structural analysis of the sugar moieties of AGPs (Figure 1). To date we have characterized several hydrolytic enzymes such as exo-β-1,3-galactanase (EC 3.2.1.145), endo-β-1,6-galactanase (EC 3.2.1.164), endo-β-1,3-galactanase (EC 3.2.1.181), β-galactosidase (EC 3.2.1.23), α-L-arabinofuranosidase (EC 3.2.1.55), and β-glucuronidase (EC 3.2.1.31).

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