12,441 research outputs found
Diverse biological effects of glycosyltransferase genes from Tartary buckwheat
Background: Tartary buckwheat (Fagopyrum tataricum) is an edible cereal crop whose sprouts have been marketed and commercialized for their higher levels of anti-oxidants, including rutin and anthocyanin. UDP-glucose flavonoid glycosyltransferases (UFGTs) play an important role in the biosynthesis of flavonoids in plants. So far, few studies are available on UFGT genes that may play a role in tartary buckwheat flavonoids biosynthesis. Here, we report on the identification and functional characterization of seven UFGTs from tartary buckwheat that are potentially involved in flavonoid biosynthesis (and have varying effects on plant growth and development when overexpressed in Arabidopsis thaliana.)
Results: Phylogenetic analysis indicated that the potential function of the seven FtUFGT proteins, FtUFGT6, FtUFGT7, FtUFGT8, FtUFGT9, FtUFGT15, FtUFGT40, and FtUFGT41, could be divided into three Arabidopsis thaliana functional subgroups that are involved in flavonoid biosynthesis of and anthocyanin accumulation. A significant positive correlation between FtUFGT8 and FtUFGT15 expression and anthocyanin accumulation capacity was observed in the tartary buckwheat seedlings after cold stress. Overexpression in Arabidopsis thaliana showed that FtUFGT8, FtUFGT15, and FtUFGT41 significantly increased the anthocyanin content in transgenic plants. Unexpectedly, overexpression of FtUFGT6, while not leading to enhanced anthocyanin accumulation, significantly enhanced the growth yield of transgenic plants. When wild-type plants have only cotyledons, most of the transgenic plants of FtUFGT6 had grown true leaves. Moreover, the growth speed of the oxFtUFGT6 transgenic plant root was also significantly faster than that of the wild type. At later growth, FtUFGT6 transgenic plants showed larger leaves, earlier twitching times and more tillers than wild type, whereas FtUFGT15 showed opposite results.
Conclusions: Seven FtUFGTs were isolated from tartary buckwheat. FtUFGT8, FtUFGT15, and FtUFGT41 can significantly increase the accumulation of total anthocyanins in transgenic plants. Furthermore, overexpression of FtUFGT6 increased the overall yield of Arabidopsis transgenic plants at all growth stages. However, FtUFGT15 shows the opposite trend at later growth stage and delays the growth speed of plants. These results suggested that the biological function of FtUFGT genes in tartary buckwheat is diverse
Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.
Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3' hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4'-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolite
Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity
Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targetsSpanish Ministerio de Economía, Industria y Competitividad , Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016‐75365‐REuropean Union’s Horizon 2020 e Marie Skłodowska‐Curie grant agreement No 84262
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Exploring the Phytochemical Landscape of the Early-Diverging Flowering Plant Amborella trichopoda Baill.
Although the evolutionary significance of the early-diverging flowering plant Amborella (Amborella trichopoda Baill.) is widely recognized, its metabolic landscape, particularly specialized metabolites, is currently underexplored. In this work, we analyzed the metabolomes of Amborella tissues using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). By matching the mass spectra of Amborella metabolites with those of authentic phytochemical standards in the publicly accessible libraries, 63, 39, and 21 compounds were tentatively identified in leaves, stems, and roots, respectively. Free amino acids, organic acids, simple sugars, cofactors, as well as abundant glycosylated and/or methylated phenolic specialized metabolites were observed in Amborella leaves. Diverse metabolites were also detected in stems and roots, including those that were not identified in leaves. To understand the biosynthesis of specialized metabolites with glycosyl and methyl modifications, families of small molecule UDP-dependent glycosyltransferases (UGTs) and O-methyltransferases (OMTs) were identified in the Amborella genome and the InterPro database based on conserved functional domains. Of the 17 phylogenetic groups of plant UGTs (A-Q) defined to date, Amborella UGTs are absent from groups B, N, and P, but they are highly abundant in group L. Among the 25 Amborella OMTs, 7 cluster with caffeoyl-coenzyme A (CCoA) OMTs involved in lignin and phenolic metabolism, whereas 18 form a clade with plant OMTs that methylate hydroxycinnamic acids, flavonoids, or alkaloids. Overall, this first report of metabolomes and candidate metabolic genes in Amborella provides a starting point to a better understanding of specialized metabolites and biosynthetic enzymes in this basal lineage of flowering plants
Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen
The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of the O antigen has been determined, but the roles of specific genes in synthesizing this structure are relatively unclear. Within the known O-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. Each ORF was mutated, and the resulting mutant lipopolysaccharide (LPS) was analyzed. Tricine SDS-PAGE revealed stepwise truncations of the O antigen that were consistent with differences in mutant LPS sugar compositions and reactivity with O-antigen-specific monoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and a model for biosynthesis of the R. etli CE3 O antigen was proposed. In this model, O-antigen biosynthesis is initiated with the addition of N-acetyl-quinovosamine-phosphate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferase WreU. Glycosyltransferases WreG, WreE, WreS, and WreT would each act once to attach mannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively. WreH would then catalyze the addition of methyl glucuronate (MeGlcA) to complete the first instance of the O-antigen repeat unit. Four subsequent repeats of this unit composed of fucose, 3OMe6dTal, and MeGlcA would be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases, WreM and WreL, along with WreH. Finally, the O antigen would be capped by attachment of di- or tri-O-methylated fucose as catalyzed by glycosyltransferase WreB
How do nematodes transfer phosphorylcholine to carbohydrates?
An unusual aspect of the biology of nematodes is the attachment of phosphorylcholine (PC) to carbohydrate. The attachment appears to play an important role in nematode development and, in some parasitic species, in immunomodulation. This article considers the nature of the biosynthetic pathway of nematode PC-containing glycoconjugates and, in particular, the identity of the final component in the pathway - the enzyme that transfers PC to carbohydrate (the 'PC transferase'). We offer the opinion that the PC transferase could be a member of the fukutin family (fukutin refers to the mutated gene product that causes Fukuyama congenital muscular dystrophy), a group of enzymes with apparent phosphoryl-ligand transferase activity that are found in organisms ranging from bacteria to humans
Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit
The volatile compounds that constitute the fruit aroma of ripe tomato (Solanum lycopersicum) are often sequestered in glycosylated form. A homology-based screen was used to identify the gene SlUGT5, which is a member of UDP-glycosyltransferase 72 family and shows specificity towards a range of substrates, including flavonoid, flavanols, hydroquinone, xenobiotics and chlorinated pollutants. SlUGT5 was shown to be expressed primarily in ripening fruit and flowers, and mapped to chromosome I in a region containing a QTL that affected the content of guaiacol and eugenol in tomato crosses. Recombinant SlUGT5 protein demonstrated significant activity towards guaiacol and eugenol, as well as benzyl alcohol and methyl salicylate; however, the highest in vitro activity and affinity was shown for hydroquinone and salicyl alcohol. NMR analysis identified isosalicin as the only product of salicyl alcohol glycosylation. Protein modelling and substrate docking analysis were used to assess the basis for the substrate specificity of SlUGT5. The analysis correctly predicted the interactions with SlUGT5 substrates, and also indicated that increased hydrogen bonding, due to the presence of a second hydrophilic group in methyl salicylate, guaiacol and hydroquinone, appeared to more favourably anchor these acceptors within the glycosylation site, leading to increased stability, higher activities and higher substrate affinities
Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme
Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl α-fluoride (4DG3αF). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3αF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl R-fluoride (kcat = 275 s-1). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3αF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.
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