Abstract
Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.
Highlights
Glycosylation regulates the activities of plant metabolites and is mediated by glycosyltransferases (GT), glycoside hydrolases (GH), and transglycosidases (TG)
The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized
Site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu169), catalytic nucleophile residues (Glu387), and His386, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH family 1 (GH1) -glucosidases that utilize a double displacement retaining mechanism
Summary
Glycosylation regulates the activities of plant metabolites and is mediated by glycosyltransferases (GT), glycoside hydrolases (GH), and transglycosidases (TG). Rice (Oryza sativa) Os9BGlu is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. Aside from oligo- and polysaccharides, glycoconjugates consist primarily of O-glycosides, glycosyl esters, S-glycosides, glycosylamines, and C-glycosyl compounds [1] These glycoconjugates may serve as active compounds themselves; as reversibly inactivated forms of phytohormones, defense compounds, monolignols, and volatiles; or as donors for acyl and glycosyl transfer reactions. The creation of glycoconjugates is primarily catalyzed by GTs, which are enzymes that transfer a sugar from a nucleotide phosphate sugar or phospholipid sugar donor to an acceptor to form a glycosidic or ester linkage [2, 7]. TGs are enzymes that transfer a sugar from a donor other than a nucleotide phosphate or phospholipid phosphate to an acceptor to form a new glycosidic linkage [2]. We show that Os9BGlu is a vacuolar TG with novel mechanistic properties that acts on a number of natural acceptors and donors, including aromatic acids and phytohormones and their glucoconjugates, suggesting a broader role for GH1 TG than previously realized
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