A UDP GLUCOSYLTRANSFERASE FROM BACILLUS-SUBTILIS SUCCESSIVELY TRANSFERS UP TO 4 GLUCOSE RESIDUES TO 1,2-DIACYLGLYCEROL - EXPRESSION OF YPFPIN ESCHERICHIA-COLI AND STRUCTURAL-ANALYSIS OF ITS REACTION-PRODUCTS

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1 967) using PCR. After cloning and expression in E. coil, SDS-PAGE show ed strong expression of a protein that had the predicted size of 43.6 kDa, Chromatographic analysis of the lipids extracted from the transfo rmed E. coil revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry, They were identified as -->6)-O-beta-D-g lucopyranosyl]-1,2-diacylglycerol, 3-[ O-beta-D-glucopyranosyl-(1-->6) -O- -->6)-O-beta-D-glucopyranosyl]-1,2-diacylgiycerol. and -->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expecte d to catalyse the synthesis of these compounds was confirmed by in vit ro assays with radioactive substrates, in these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-gluco pyranosyl]-1,2-diacylglycerol, which was not detected in the lipid ext ract of transformed cells, Experiments with some of the above-describe d glycolipids as C-14-labelled sugar accepters and unlabelled UDP-gluc ose as glucose donor suggest that the ypfP gene codes for a new proces sive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. Th is glucosyltransferase can use diacylglycerol, noglucosyl-diacylglycer ol,diglucosyldiacylglycerol or triglucosyl-diacylglycerol as sugar acc eptor, which, apart from the first member, are formed by repetitive ad dition of a glucopyranosyl residue in beta (1-->6) linkage to the prod uct of the preceding reaction.