Purification and characterization of a novel glucosyltransferase specific to 27beta-hydroxy steroidal lactones from Withania somnifera and its role in stress responses.
Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27beta-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with beta-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an alpha-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3beta-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.
Madina
Bhaskara Reddy
BR
National Botanical Research Institute, Rana Pratap Marg, Lucknow, U.P-226001, India.
Sharma
Lokendra Kumar
LK
Chaturvedi
Pankaj
P
Sangwan
Rajender Singh
RS
Tuli
Rakesh
R
D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D010449:Peptide Mapping; D010935:Plant Diseases; D018515:Plant Leaves / Q000201:enzymology; D020156:Salicylic Acid / Q000494:pharmacology; D013379:Substrate Specificity; D032341:Withania / Q000187:drug effects / Q000201:enzymology*
C036655:1,3-alpha-D-glucan synthase; D005964:Glucosyltransferases; D020156:Salicylic Acid; D004875:Ergosterol