title: Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities.
abstract: 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-beta-D-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-beta-D-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->3)-O-beta-D-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-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.
authors.affiliation: Biozentrum Klein Flottbek, University of Hamburg, Hamburg 22609, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Hölzl
authors.forename: Martina
authors.initials: M
authors.lastname: Leipelt
authors.forename: Claudia
authors.initials: C
authors.lastname: Ott
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zähringer
authors.forename: Buko
authors.initials: B
authors.lastname: Lindner
authors.forename: Dirk
authors.initials: D
authors.lastname: Warnecke
authors.forename: Ernst
authors.initials: E
authors.lastname: Heinz
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006017:Glycolipids / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D006017:Glycolipids; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase