Principal Investigator: Takeshi Ishimizu
Grant Title: Identification of Glycosyltransferases Involved in Pectin Biosynthesis
AbstractPectin, a component of plant cell walls, is composed of three regions:homogalacturonan, rhamnogalacturonan I (RG-I), and RG-II.
Author’s
Approximately 30 glycosyltransferases are thought to be involved inPhotographtheir biosynthesis, but few have been identified. Therefore, the role(Optional)
and function of pectin remain largely unexplored. Our group hasSize is notpreviously identified genes for rhamnosyltransferase involved in RG-Ispecified.
biosynthesis (Takenaka et al., 2018). Based on this experience, inorder to elucidate the function of pectin, we started the study toidentify the genes of two enzymes, RG-II apiosyltransferase and RG-I galactosyltransferase. Because
the genetic approach for identification of genes encoding pectin-biosynthetic glycosyltransferases
has not been successful, we attempted to identify these genes by biochemical approaches.
RG-II apiosyltransferase is a critical enzyme that synthesizes RG-II side chain. The apiose
residue of RG-II forms a boron diester bond and is involved in the dimerization of RG-II. Thisapiosyltransferase activity has never been detected so far, because no preparation method for its
donor substrate, UDP-apiose, has been established. UDP-apiose is unstable because the hydroxyl
group at the second position of the sugar of UDP-apiose is located close to the adjacent phosphateester and can easily be nucleophilic attacked to form cyclic phosphate. In this study, the stabilizationof UDP-apiose was achieved by orienting triethylamine as a counter ion. We prepared UDP-apiosewith sufficient amount for using RG-II apiosyltransferase assay. The results were published in thejournal Carbohydrate Research (Fujimori et al., 2019). Currently, the gene encoding this enzyme issearched from Arabidopsis genome using this biochemical method.
RG-I galactosyltransferase is a key enzyme in the formation of RG-I side chain galactans, whichinteract with cellulose in the cell wall and are thought to play a role in the firmness and softness ofplants. In addition, RG-I with galactan has been shown to be an effective medicinal component withimmune-enhancing activity in Chinese herbal medicine. In this study, an acceptor substrate for thisenzyme was prepared and an assay method for this enzyme was constructed using the substrate. TheRG-I main chain of oligosaccharides of a certain length (>10 sugars) was found to be the best
substrate for this enzyme. We also found that this enzyme was superactivated in the presence ofcertain cationic surfactants and polyelectrolytes. This would suggest that this enzyme has ahydrophobic region and interacts with other proteins. The results were published in the journal Plant
Physiology and Biochemistry (Matsumoto et al., 2019). As this is the first time that this enzyme
activity has been detected, we have applied to Enzyme Nomenclature to issue an enzyme number(EC number). Using this established method for measuring the activity of this enzyme, we aresearching for the gene encoding this enzyme. The identification of these genes will lead toelucidation of the role and function of pectin.
References
1. Takenaka et al. (2018) Nature Plants 4, 669-676.
2. Fujimori et al. (2019) Carbohydr. Res. 477, 20-25.
3. Matsumoto et al. (2019) Plant Physiol. Biochem. 142, 173-178.