Glucosyl- and Acetyltransferases Involved in the Biosynthesis of Glycolipids from Candida bogoriensis

Abstract

Abstract 1. Crude extracts prepared by sonicating Candida bogoriensis cells contained glucosyltransferases catalyzing the transfer of glucose from UDP-[14C]glucose to 13-hydroxydocosanoic acid (HDA) (transferase 1) and methyl 13-glucopyranosyloxydocosanoate (methyl GlcHDA) (transferase 2) when these lipids were added as acceptors. The enzymatic products were characterized by thin layer chromatographic and gas-liquid chromatographic comparison to derivatives of GlcHDA and 13-glucopyranosylglucopyranosyloxydocosanoate (Glc2HDA) prepared from cultures of C. bogoriensis. 2. The transferases were purified 20- and 30-fold, respectively, with no separation of the two activities and with a constant ratio of the two activities obtained in successive peak fractions from a DEAE-Sephadex column, a hydroxyl-apatite column, a gel filtration column, and a disc gel electrophoresis fractionation. 3. Both transferases were specific for UDP-glucose, showing apparent Km values of 104 and 88 µm, respectively, for this compound. 4. Glucosyltransferase 1 showed 4- to 5-fold greater activity with HDA than with its methyl ester. A series of hydroxystearate isomers also showed acceptor activity. The activity with 9-hydroxystearate was 60% of that with HDA, whereas the acceptor activity decreased as the hydroxyl group was moved toward either the methyl or carboxyl end of the fatty acid chain. 5. Glucosyltransferase 2 exhibited severe substrate inhibition with GlcHDA as acceptor and a much higher reaction velocity when the methyl ester of GlcHDA was utilized as acceptor. 6. Crude extracts of C. bogoriensis also contained acetyltransferase(s) catalyzing incorporation of [14C]acetate from [14C]acetyl coenzyme A into Ac2Glc2HDA (the diacetate of Glc2HDA) characterized by thin layer chromatographic comparison with the glycolipid isolated from C. bogoriensis cultures. 7. Acetone extraction of the lyophilized extracts, followed by DEAE-Sephadex chromatography, freed the acetyl-transferase(s) of endogenous acceptor, making activity dependent upon added methyl Glc2HDA or its monoacetate as acceptor. Methyl GlcHDA was a poor acceptor for the acetyl groups. 8. In fermentor cultures the peak of glucosyltransferase 1 and 2 activities was reached in 1½ days and the peak of acetyltransferase activity was reached in 3 days. This time course is consistent with involvement of these enzymes in the biosynthesis of extracellular Ac2Glc2HDA, and with the tentative conclusion that extracellular AcGlc2HDA and Glc2HDA were formed by deacetylation of Ac2Glc2HDA.