Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities

• Published in: Glycobiology (Oxford) Vol. 15; no. 9; pp. 874 - 886
• Main Authors: Hölzl, Georg, Leipelt, Martina, Ott, Claudia, Zähringer, Ulrich, Lindner, Buko, Warnecke, Dirk, Heinz, Ernst
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.09.2005; Oxford Publishing Limited (England)
• Subjects: 3-diazolo)-hexanoyl]-ceramide; Agrobacterium tumefaciens; Agrobacterium tumefaciens - enzymology; Agrobacterium tumefaciens - genetics; Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; combined gas–liquid chromatography/mass spectrometry analysis; d-erythro-N [6-amino-N-4′(7-nitrobenzo-2-oxa-1; DAG; diacylglycerol(s); diacylglycerols; electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry; enzyme activity; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; ESI FT-ICR MS; galactose; galactosyl diacylglycerol; galactosyltransferase; GCS; Gene Expression; genetically engineered microorganisms; GLC-MS; glucosylceramide; glucosylceramide synthase; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; glycolipids; Glycolipids - genetics; Glycolipids - metabolism; glycosyltransferase family 21; GT21; hexosyltransferases; mass spectrometry; Mesorhizobium loti; Molecular Sequence Data; NBD-Cer; NMR; nuclear magnetic resonance; open reading frame; ORF; PCR; Pichia - enzymology; Pichia - genetics; Pichia pastoris; polymerase chain reaction; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Species Specificity; Substrate Specificity - physiology; tetrahydrofuran; TGD; THF; thin layer chromatography; TLC; triglycosyl diacylglycerol; UDP-Gal; UDP-Glc; uridine; uridine diphosphogalactose; uridine diphosphoglucose
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwi066

The glycosyltransferase family 21 (GT21) includes both enzymes of eukaryotic and prokaryotic organisms. Many of the eukaryotic enzymes from animal, plant, and fungal origin have been characterized as uridine diphosphoglucose (UDP-Glc):ceramide glucosyltransferases (glucosylceramide synthases [Gcs], EC 2.4.1.80). As the acceptor molecule ceramide is not present in most bacteria, the enzymatic specificities and functions of the corresponding bacterial glycosyltransferases remain elusive. In this study, we investigated the homologous and heterologous expression of GT21 enzymes from Agrobacterium tumefaciens and Mesorhizobium loti in A. tumefaciens, Escherichia coli, and the yeast Pichia pastoris. Glycolipid analyses of the transgenic organisms revealed that the bacterial glycosyltransferases are involved in the synthesis of mono-, di- and even tri-glycosylated glycolipids. As products resulting from their activity, we identified 1,2-diacyl-3-(O-b-d-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-b-d-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-b-d-galacto-pyranosyl-(1[rightwards arrow]6)-O-b-d-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-b-d-gluco-pyranosyl-(1[rightwards arrow]6)-O-b-d-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-b-d-gluco-pyranosyl-(1[rightwards arrow]6)-O-b-d-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-b-d-gluco-pyranosyl-(1[rightwards arrow]3)-O-b-d-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-b-d-galacto-pyranosyl-(1[rightwards arrow]6)-O-b-d-galacto-pyranosyl-(1[rightwards arrow]6)-O-b-d-galacto-pyranosyl]-sn-glycerol could be separated in a pure form. In vitro enzyme assays showed that the glycosyltransferase from A. tumefaciens favours uridine diphosphogalactose (UDP-Gal) over UDP-Glc. In conclusion, the bacterial GT21 enzymes differ from the eukaryotic ceramide glucosyltransferases by the successive transfer of up to three galactosyl and glucosyl moieties to diacylglycerol.