Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols

Abstract

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis[Jorasch, P., Wolter, F.P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol.29, 419–430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin‐layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyrano‐syl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl]‐sn‐glycerol, sn‐3′‐[O‐β‐d‐glucopyranosyl]‐phosphatidylglycerol and sn‐3′‐[O‐(6′″‐O‐acyl)‐β‐d‐glucopyranosyl‐(1′′′→6′′)‐O‐β‐d‐glucopyranosyl]‐sn‐2′‐acyl‐phospha‐tidylglycerol. A 1,2‐diacyl‐3‐[O‐β‐d‐galactopyranosyl]‐sn‐glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as ¹⁴C‐labeled sugar acceptors and unlabeled UDP‐glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP‐glucose:1,2‐diacylglycerol‐3‐β‐d‐glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.