Cloning and expression of solanidine UDP‐glucose glucosyltransferase from potato

Moehs, Charles P.; Allen, Patrick; Friedman, Mendel; Belknap, William R.

doi:10.1046/j.1365-313x.1997.11020227.x

Cited by 151 publications

(112 citation statements)

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“…B, sinapyl alcohol was used as substrate in the assay. rified from a wide range of different plant families (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), there have been no previous attempts to study this multigene family in a single species and therefore no possibility of directly comparing relative activities across family members. Access to the kinetic analysis of many family members from a single species can provide increased confidence of the range of substrates that may be used by each of these enzymes in vitro and thereby a foundation for exploring their substrates in vivo and their physiological roles in the plant.…”

Section: Discussionmentioning

confidence: 99%

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

Lim

Parr

et al. 2001

Journal of Biological Chemistry

197

161

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Sinapic acid is a major phenylpropanoid in Brassicaceae providing intermediates in two distinct metabolic pathways leading to sinapoyl esters and lignin synthesis. Glucosyltransferases play key roles in the formation of these intermediates, either through the production of the high energy compound 1-O-sinapoylglucose leading to sinapoylmalate and sinapoylcholine or through the production of sinapyl alcohol-4-O-glucoside, potentially leading to the syringyl units found in lignins. While the importance of these glucosyltransferases has been recognized for more than 20 years, their corresponding genes have not been identified. Combining sequence information in the Arabidopsis genomic data base with biochemical data from screening the activity of recombinant proteins in vitro, we have now identified five gene sequences encoding enzymes that can glucosylate sinapic acid, sinapyl alcohol, and their related phenylpropanoids. The data provide a foundation for future understanding and manipulation of sinapate metabolism and lignin biology in Arabidopsis.

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Section: Discussionmentioning

confidence: 99%

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

Lim

Parr

et al. 2001

Journal of Biological Chemistry

197

161

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“…1 corresponds to a putative steroid-binding domain found in a solanidine glucosyltransferase from potato (see Ref. 67, GenBank TM accession number U82367) and mammalian steroid UDP-glucuronosyltransferases (e.g. UGT2B7, GenBank TM accession number J05428).…”

Section: Isolation and Cloning Of Putative Sterol Glycosyltransferasementioning

confidence: 99%

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Warnecke¹,

Erdmann²,

Fahl³

et al. 1999

Journal of Biological Chemistry

122

135

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Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose: sterol glucosyltransferase (EC 2.4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 ‫؍‬ YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-␤-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-␤-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.Sterol glycosides are widespread membrane lipids, occurring in all plants, several algae (1-3), some fungi (4 -9), slime molds (10 -12), Dictyostelium (13), a few bacteria (14 -19), and even animals (20 -23). The knowledge base for sterol glycosides is rather limited compared with free sterols and sterol esters, where the synthesis, transport, and functions have been studied extensively in animals (24 -29), plants, (30 -35), and yeast (36 -40). The basis for studies on the functions of sterol glycosides is the assumption that free sterols and sterol glycosides differ physiologically. It is obvious that the attachment of a glycosyl moiety to the sterol backbone alters the physical properties of this lipid. As a result, there are changes in the properties of membranes containing different proportions of free sterols and sterol glycosides. Such changes have been studied with artificial membranes in terms of membrane fluidity, permeability, hydration, and phase behavior (41)(42)(43)(44). However, we still do not know how free sterols and sterol glycosides differ physiologically in biological membranes and why many eukaryotic organisms synthesize sterol glycosides. One of the main reasons for our limited knowledge in this field is the lack of a genetic approach. The objective of the present work was the isolation and characterization of sterol glycosyltransferase genes from eukaryotic organisms. We expect that genetic manipulation of these genes will facilitate the elucidation of sterol glycoside functions in these organisms.The predominating sugar moiety in sterol glycosides is glucose. Besides plants...

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“…Subsequently, related 4 genes for enzymes that mainly catalyzed the glucosylation of flavonols in the biosynthesis of anthocyanins were cloned from many plant species using the gene for UFGT from maize as probe (for review, [8]). Other genes for GTase-related enzymes that catalyze the glucosylation of a variety of low-molecular-mass compounds have been isolated from plants: the gene for an IAA glucosyltransferase from maize [3]; the gene for a solanidine glucosyltransferase from potato [9]; and some genes for GTase-homologous enzymes that are expressed during the ripening of fruit [10], in response to wounding or in response to treatment with salicylic acid [11]. Much further work is needed to clarify the relationships between the structures and functions of GTases and to understand their roles in plants.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells

et al. 2000

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The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1,200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%.Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn 2+ , Co 2+ and Cu 2+ ions, as well as by SH-blocking reagents. The Km values for UDP-glucose, scopoletin and esculetin were 43 µM, 150 µM and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

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Cloning and expression of solanidine UDP‐glucose glucosyltransferase from potato

Cited by 151 publications

References 0 publications

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells

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