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

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 Glc2H