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J Biol Chem, Vol. 274, Issue 19, 13048-13059, May 7, 1999

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

Dirk WarneckeDagger , Ralf Erdmann, Annette FahlDagger , Bernhard HubeDagger , Frank MüllerDagger , Thorsten ZankDagger , Ulrich Zähringerparallel , and Ernst HeinzDagger

From the Dagger  Universität Hamburg, Institut für Allgemeine Botanik, 22609 Hamburg,  Freie Universität Berlin, Institut für Biochemie, Limonenstrasse 7, 12203 Berlin, and parallel  Forschungszentrum Borstel, 23845 Borstel, Germany

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-beta -D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta -D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.


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