A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2‐diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products

Jorasch, Petra; Wolter, Frank P.; Zähringer, Ulrich; Heinz, Ernst

doi:10.1046/j.1365-2958.1998.00930.x

Cited by 137 publications

(135 citation statements)

References 41 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…All other hexose ring protons showed high coupling constant values (J 2, 3 9.5, J 3, 4 9.6, and J 4, 5 9.8) characteristic for the gluco configuration in a pyranoside that was further supported by the chemical shift data (Table III). These data are in full agreement with a terminal tetra-O-acetyl ␤-glucopyranose (68).…”

Section: The Molecular Ion [M ϫ H]supporting

confidence: 87%

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Warnecke¹,

Erdmann²,

Fahl³

et al. 1999

Journal of Biological Chemistry

Self Cite

122

135

View full text Add to dashboard Cite

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose: sterol glucosyltransferase (EC 2.4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 ‫؍‬ YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-␤-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-␤-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.Sterol glycosides are widespread membrane lipids, occurring in all plants, several algae (1-3), some fungi (4 -9), slime molds (10 -12), Dictyostelium (13), a few bacteria (14 -19), and even animals (20 -23). The knowledge base for sterol glycosides is rather limited compared with free sterols and sterol esters, where the synthesis, transport, and functions have been studied extensively in animals (24 -29), plants, (30 -35), and yeast (36 -40). The basis for studies on the functions of sterol glycosides is the assumption that free sterols and sterol glycosides differ physiologically. It is obvious that the attachment of a glycosyl moiety to the sterol backbone alters the physical properties of this lipid. As a result, there are changes in the properties of membranes containing different proportions of free sterols and sterol glycosides. Such changes have been studied with artificial membranes in terms of membrane fluidity, permeability, hydration, and phase behavior (41)(42)(43)(44). However, we still do not know how free sterols and sterol glycosides differ physiologically in biological membranes and why many eukaryotic organisms synthesize sterol glycosides. One of the main reasons for our limited knowledge in this field is the lack of a genetic approach. The objective of the present work was the isolation and characterization of sterol glycosyltransferase genes from eukaryotic organisms. We expect that genetic manipulation of these genes will facilitate the elucidation of sterol glycoside functions in these organisms.The predominating sugar moiety in sterol glycosides is glucose. Besides plants...

show abstract

Section: The Molecular Ion [M ϫ H]supporting

confidence: 87%

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Warnecke¹,

Erdmann²,

Fahl³

et al. 1999

Journal of Biological Chemistry

Self Cite

122

135

View full text Add to dashboard Cite

show abstract

“…1). For this purpose, glycosyltransferases were selected from bacterial origin that had previously been characterized by heterologous expression in prokaryotic or eukaryotic hosts (Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Synechococcus) and subsequent analysis of newly formed glycolipids (20)(21)(22)(23)(24)(25)(26). Additional putative glycosyltransferases were identified in bacteria based on sequence similarity to known genes (Table 1) (26), and by this 20 bacterial genes were selected for expression in plants.…”

Section: Resultsmentioning

confidence: 99%

Functional differences between galactolipids and glucolipids revealed in photosynthesis of higher plants

Hölzl

Witt

Kelly

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Galactolipids represent the most abundant lipid class in thylakoid membranes, where oxygenic photosynthesis is performed. The identification of galactolipids at specific sites within photosynthetic complexes by x-ray crystallography implies specific roles for galactolipids during photosynthetic electron transport. The preference for galactose and not for the more abundant sugar glucose in thylakoid lipids and their specific roles in photosynthesis are not understood. Introduction of a bacterial glucosyltransferase from Chloroflexus aurantiacus into the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana resulted in the accumulation of a glucose-containing lipid in the thylakoids. At the same time, the growth defect of the dgd1 mutant was complemented. However, the degree of trimerization of light-harvesting complex II and the photosynthetic quantum yield of transformed dgd1 plants were only partially restored. These results indicate that specific interactions of the galactolipid head group with photosynthetic protein complexes might explain the preference for galactose in thylakoid lipids of higher plants. Therefore, galactose in thylakoid lipids can be exchanged with glucose without severe effects on growth, but the presence of galactose is crucial to maintain maximal photosynthetic efficiency.chloroplast ͉ galactose ͉ glucose ͉ lipid ͉ glucosylgalactosyldiacylglycerol D irectly or indirectly, almost all life on earth depends on the photosynthetic conversion of water, CO 2 , and sunlight into chemical energy and oxygen. In cyanobacteria, green algae, and plants, the primary reactions of oxygenic photosynthesis are executed in thylakoid membranes. They harbor a set of multimeric protein complexes embedded into a lipid matrix of unique composition. The structure of the protein complexes and the lipid composition of thylakoid membranes are highly conserved in all organisms performing oxygenic photosynthesis. Thylakoid lipids comprise the two galactolipids monogalactosyldiacylglycerol (␤GalDG) and digalactosyldiacylglycerol (␣Gal␤GalDG), a sulfolipid, and phosphatidylglycerol as the only phospholipid (1-3). Based on their high proportion in thylakoid membranes and the abundance of plants and algae, galactolipids represent the most abundant lipid class in the biosphere (4, 5). Galactolipids are crucial to establish the proton-and ion-impermeable matrix of chloroplast membranes. An appropriate ratio of ␤GalDG to ␣Gal␤GalDG is required to maintain the intricate bilayer characteristics required for insertion, folding, movement, and conformational changes of membrane proteins (6). Crystalline chlorophyll-protein complexes contain galactolipid molecules firmly bound to specific sites, some of them in close proximity to the electron transfer chain. This association implies important roles of galactolipids in photosynthetic exciton and electron transfer within and between the different complexes (7-9). Mutants and transgenic plants of Arabidopsis thaliana are the basis for our current understanding of the role of th...

show abstract

“…To date, identification of two glucosyldiacylglycerol synthases has been reported, namely, ypfP of B. subtilis and MGlcDAG synthase of A. laidlawii (Jorasch et al, 1998;Berg et al, 2001). However, neither of these have significant sequence similarity to MGlcDG synthase of cyanobacteria.…”

Section: Discussionmentioning

confidence: 99%

“…(2) The enzyme should not have a high overall similarity to proteins that have a well-known function. There are two reports of glucosyltransferase genes encoding for the enzymes that synthesize mono-or polyglucosyldiacylglycerol (Jorasch et al, 1998;Berg et al, 2001). However, no homolog has been found in any cyanobacterial genome other than SQDG synthase from Synechocystis (Berg et al, 2001).…”

Section: Comparative Genomic Analysis Found Candidates For Mglcdg Synmentioning

confidence: 99%

“…In Bacillus subtilis, MGlcDG is about 10% of the total lipid (Matsumoto et al, 1998). Although the genes for the MGlcDG synthases of these bacteria have been cloned (Jorasch et al, 1998;Berg et al, 2001), again there is no ortholog in any cyanobacterial genome. Thus, to date, the gene for MGlcDG synthase is unknown in cyanobacteria even though the first genome sequence of the bacteria was completed almost a decade ago (Kaneko et al, 1996).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Comparative Genomic Analysis Revealed a Gene for Monoglucosyldiacylglycerol Synthase, an Enzyme for Photosynthetic Membrane Lipid Synthesis in Cyanobacteria

Awai

Kakimoto²,

Awai³

et al. 2006

Plant Physiology

View full text Add to dashboard Cite

Cyanobacteria have a thylakoid lipid composition very similar to that of plant chloroplasts, yet cyanobacteria are proposed to synthesize monogalactosyldiacylglycerol (MGDG), a major membrane polar lipid in photosynthetic membranes, by a different pathway. In addition, plant MGDG synthase has been cloned, but no ortholog has been reported in cyanobacterial genomes. We report here identification of the gene for monoglucosyldiacylglycerol (MGlcDG) synthase, which catalyzes the first step of galactolipid synthesis in cyanobacteria. Using comparative genomic analysis, candidates for the gene were selected based on the criteria that the enzyme activity is conserved between two species of cyanobacteria (unicellular [Synechocystis sp. PCC 6803] and filamentous [Anabaena sp. PCC 7120]), and we assumed three characteristics of the enzyme; namely, it harbors a glycosyltransferase motif, falls into a category of genes with unknown function, and shares significant similarity in amino acid sequence between these two cyanobacteria. By a motif search of all genes of Synechocystis, BLAST searches, and similarity searches between these two cyanobacteria, we identified four candidates for the enzyme that have all the characteristics we predicted. When expressed in Escherichia coli, one of the Synechocystis candidate proteins showed MGlcDG synthase activity in a UDP-glucose-dependent manner. The ortholog in Anabaena also showed the same activity. The enzyme was predicted to require a divalent cation for its activity, and this was confirmed by biochemical analysis. The MGlcDG synthase and the plant MGDG synthase shared low similarity, supporting the presumption that cyanobacteria and plants utilize different pathways to synthesize MGDG.

show abstract

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2‐diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products

Cited by 137 publications

References 41 publications

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Cloning and Functional Expression of UGT Genes Encoding Sterol Glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Functional differences between galactolipids and glucolipids revealed in photosynthesis of higher plants

Comparative Genomic Analysis Revealed a Gene for Monoglucosyldiacylglycerol Synthase, an Enzyme for Photosynthetic Membrane Lipid Synthesis in Cyanobacteria

Contact Info

Product

Resources

About