Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum

Wojciechowski, Zdzisław A.; Zimowski, Jan; Zimowski, Janusz; Łyżnik, Anna

doi:10.1016/0005-2744(79)90156-6

Cited by 36 publications

(12 citation statements)

References 16 publications

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“…The complete lack of SaGT4A activity towards digitoxigenin and sarsasapogenin, in contrast to all other steroids tested, is, most probably, resulting from b configuration of the H atom at C-5 in these compounds. A cis-coupling of rings A and B present in 5b-H steroids results in a non-planer steroid nucleus, in contrast to 5a-H or 5-en compounds which have much more planer ring system (Wojciechowski et al, 1979). The axial orientation of the C-25 methyl group present in sarsasapogenin would also be incompatible for reactions with this enzyme.…”

Section: Discussionmentioning

confidence: 99%

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum

et al. 2005

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Steroidal saponins are widely distributed in many plant species. Their diverse structures have resulted in a wide range of applications, including drug and medicine production. It has been suggested that the nature of the non-saccharide and oligosaccharide portions of the saponin molecule both contribute to the properties of individual saponins. Despite numerous studies on the occurrence, chemical structure, and varying pharmaceutical activities of steroidal saponins, their biosynthesis pathway is poorly understood. Glycosylation is thought to be the final step in steroidal saponin biosynthesis and it is thought to be involved in regulating the biological activities of saponins. Isolation of the glycosyltransferases that catalyze the transfer of sugar molecules to steroidal compounds will help to clarify the mechanisms that produce diverse saponins and control their activities in plants. In this study, we obtained three cDNAs encoding putative glycosyltransferases from Solanum aculeatissimum. One of the three, SaGT4A showed UDP-glucosyltransferase activity. This is the first cloned glucosyltransferase involved in steroidal saponin biosynthesis. SaGT4A catalyzes the 3-O-glucosylation of steroidal sapogenins, such as diosgenin, nuatigenin, and tigogenin. This enzyme also glucosylates steroidal alkaloids, such as solanidine, solasodine, and tomatidine. Gene expression analysis revealed that the accumulation of SaGT4A transcripts showed a unique response to wounding stress indicating the involvement of SaGT4A in plant defense system.

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Section: Discussionmentioning

confidence: 99%

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum

et al. 2005

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“…We also examined the nucleotide specificity of GCS and found that, surprisingly, GCS is able to efficiently utilize CDP-Glc and TDP-Glc as glucose donors (see Table IV). While CDP-Glc and TDP-Glc are not naturally occurring glucose donors, earlier studies have also described UDP-Glc:glucosyltransferases that are able to utilize CDP-Glc and TDP-Glc (54,55).…”

Section: Table IIImentioning

confidence: 99%

Purification and Characterization of UDP-glucose:Ceramide Glucosyltransferase from Rat Liver Golgi Membranes

Paul

Kamisaka²,

Marks

et al. 1996

Journal of Biological Chemistry

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We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)) from rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment >10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, 125 I labeling, and SDS-polyacrylamide gel electrophoresis, which demonstrated that two polypeptides (60 -70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-sphingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.

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“…Besides plants, UDP-glucose:sterol glucosyltransferase activity was determined by in vitro enzyme assays in Saccharomyces cerevisiae (45)(46)(47)(48), Candida bogoriensis (49), other fungi (50), and Physarum polycephalum (51,11). Although the lipid composition of S. cerevisiae was studied in detail during the last decades, there are only rare reports on the actual isolation of sterol glucoside (SG) 1 from this yeast (8,9).…”

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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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Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum

Cited by 36 publications

References 16 publications

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum

Purification and Characterization of UDP-glucose:Ceramide Glucosyltransferase from Rat Liver Golgi Membranes

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

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