Purification and physico-kinetic characterization of 3β-hydroxy specific sterol glucosyltransferase from Withania somnifera (L) and its stress response Bhaskara Reddy Madina a , Lokendra Kumar Sharma a , Pankaj Chaturvedi a , Rajender Singh Sangwan b , Rakesh Tuli a, ⁎ a National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, (U.P.) India b Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India Received 12 May 2006; received in revised form 25 December 2006; accepted 26 December 2006 Available online 8 January 2007 Abstract Sterol glycosyltransferases catalyze the synthesis of diverse glycosteroids in plants, leading to a change in their participation in cellular metabolism. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a cytosolic sterol glucosyltransferase was purified 3406 fold to near homogeneity from W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme was active with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols and phytosterols with 3β-OH group. It showed a low level of activity with flavonoids and isoflavonoids. The enzyme gave maximum K cat /K m value (0.957) for 24-methylenecholesterol that resembles aglycone structure of pharmacologically important sitoindosides VII and VIII from W. somnifera. The enzyme follows ordered sequential bisubstrate mechanism of reaction, in which UDP-glucose and sterol are the first and second binding substrates. This is the first detailed kinetic study on purified plant cytosolic sterol glucosyltransferases. Results on peptide mass fingerprinting and substrate specificity suggested that the enzyme belongs to the family of secondary metabolite glucosylating glucosyltransferases. The enzyme activity exhibited a rapid in vivo response to high temperature and salicylic acid treatment of plants, suggesting its physiological role in abiotic and biotic stress. © 2007 Elsevier B.V. All rights reserved. Keywords: Heat stress; Salicylic acid signal; Sterol glucoside; Sterol glucosyltransferase; Stress response; Substrate specificity; Withanosides 1. Introduction Enzymatic glycosylation involves the transfer of sugar from an activated donor (nucleotidediphosphate sugar) to an aglycone substrate. Sterol glycosyltransferases in plants cata- lyze glycosylation of phytosterols and related compounds to generate their glyco-conjugates. Sterol glycosyltransferases play an important regulatory role in the activity of sterols in higher organisms ranging from molds and plants to insects and mammals [1–3]. A group of oxidized sterols, called brassinos- terols, function as plant growth regulators and influence growth and development in plants [4]. Mutations in sterol transforma- tion pathway deviates the normal balance of different sterol metabolites and lead to defects in embryonic and post- embryonic development and flower morphogenesis [5]. In plant cells, sterols are synthesized primarily in endoplas- mic reticulum using mevalonate pathway of isoprenogenesis by generating prenyl precursors from cytosol. Some contribution of the plastid localized DOXP pathway of isoprenogenesis has also been suggested [6]. These isoprene units lead to the biosynthesis of 2,3-oxidosqualene, which serves as the common progenitor of different classes of sterols. Most of the higher plant sterols possess β-OH group at C-3 position and largely occur in free form. However, some of them are structurally diversified through a variety of catalytic transformations including desaturation, chain-elongation, cyclisation, esterifica- tion, epoxidation, hydroxylation and glycosylation. Amongst Biochimica et Biophysica Acta 1774 (2007) 392 – 402 www.elsevier.com/locate/bbapap ⁎ Corresponding author. Tel./fax: +91 522 2205698 (Direct); tel.: +91 522 2205849x324 (Off), +91 522 2789499, 2789535 (Res.); fax: +91 522 2205836, 2205839. E-mail address: rakeshtuli@hotmail.com (R. Tuli). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.12.009