Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase fromGymnema sylvestreR.Br. Catalyzing Biosynthesis of Steryl Glucosides

Abstract

Gymnema sylvestre R.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed in Escherichia coli and biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene from G. sylvestre R.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

Identification and functional characterization of DzS3GT, a cytoplasmic glycosyltransferase catalyzing biosynthesis of diosgenin 3-O-glucoside in Dioscorea zingiberensis

“…The optimal glycosyltransferase activity of rDzS3GT was obtained at 50 °C and at pH 6.5, higher than the optimum temperature of 30 °C and pH 6.5 previously reported for a eukaryotic SGT protein from Micromonospora rhodorangea (Huu et al 2016), which may be caused by the different posttranslational modifications of the SGT protein. The optimal pH for rDzS3GT activity was slightly acidic and close to neutral, similar to the reported rGsSGT (Tiwari et al 2014).…”

“…This expression pattern is obviously different from tissue profiles of SGT genes from other plant species, such as G. sylvestre (Tiwari et al 2014) and S. aculeatissimum (Kohara et al 2005). Furthermore, higher DzS3GT transcript level and DzS3GT activity in the leaves suggests that trillin is mainly synthesized in the leaves of D. zingiberensis, and other plant organs might also contribute to trillin biosynthesis.…”

“…SGT belongs to family 1 of a classification scheme that contains 94 GT families described in CAZY (carbohydrate active enzymes) database (http://www.cazy.org/) and usually exhibits broad substrate specificity (Cantarel et al 2009). GsSGT from Gymnema sylvestre can glycosylate different kinds of sterols, such as cholesterol, ergosterol and stigmasterol (Tiwari et al 2014). SaGT4A from Solanum aculeatissimum can glycosylate both steroidal sapogenins and steroidal alkaloids (Kohara et al 2005).…”

“…SaGT4A from Solanum aculeatissimum can glycosylate both steroidal sapogenins and steroidal alkaloids (Kohara et al 2005). S3GTs have been functionally characterized at the molecular level in several plant species till now, including Avena sativa (Warnecke et al 1997), Solanum aculeatissimum (Kohara et al 2005), Withania somnifera , Arabidopsis thaliana (Debolt et al 2009) and Gymnema sylvestre (Tiwari et al 2014). Four full-length SGTs have been cloned using rapid amplification of cDNA ends (RACE) method.…”

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

“…In the first reaction, the sugar moiety from nucleotide-activated glycosyl donors was attached to steroids at different positions, most commonly at the C-3 hydroxyl group (OH), under the action of nucleotide dependent SGTs 15,16 . The glycosylation of a hydroxyl group at the C-3 position of steroids was well characterized and a few of steroidal 3β-glucosyltransferases were isolated from diverse species 17,18 . However, the reports of SGTs specific for positions other than C-3 of steroids are limited.…”