Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases

Sheikh, M. Osman; Halmo, Stephanie M.; Patel, Sneha; Middleton, Dustin R.; Takeuchi, Hideyuki; Schafer, Christopher M.; West, Christopher M.; Haltiwanger, Robert S.; Avci, Fikri Y.; Moremen, Kelley W.; Wells, Lance

doi:10.1093/glycob/cww114

Cited by 45 publications

(45 citation statements)

References 37 publications

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“…In the initial screening, each reaction mixture (25 μl in total) contained Tris–HCl buffer (50 mM, pH 7.5, 10% glycerol, and 10‐mM 2‐mercaptoethanol), 250‐mM UDP‐glucose, alcohol substrates (2 μl of a 10‐mM stock solution), and purified protein (2–5 μg per reaction) according to Song, Zhao, et al () and Sheikh et al (). The reaction mixture was incubated at 30°C for 30 min in independent wells of a white, flat bottom 96‐well assay plate (Corning) and allowed to incubate at ambient temperature.…”

Section: Methodsmentioning

confidence: 99%

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Jing

Zhang

Gao

et al. 2018

Plant Cell & Environment

121

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Plants emit a variety of volatiles in response to herbivore attack, and (Z)‐3‐hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)‐3‐hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)‐3‐hexenol remained unknown until today. In this study, uridine‐diphosphate‐dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)‐3‐hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53‐1 and UGT85A53‐2 showed the highest activity towards (Z)‐3‐hexenol and are distinct from UGT85A53‐3, which displayed a similar catalytic efficiency for (Z)‐3‐hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)‐3‐hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53‐1 in tobacco significantly increased the level of (Z)‐3‐hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)‐3‐hexenol UGT not only provides the foundation for the biotechnological production of (Z)‐3‐hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.

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Section: Methodsmentioning

confidence: 99%

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Jing

Zhang

Gao

et al. 2018

Plant Cell & Environment

121

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“…Each reaction mixture (5 ml in total) contained 50 mM Tris-HCl buffer (pH 7.5, 10% glycerol, and 10 mM 2-mercaptoethanol), 250 mM UDP-glucose, alcohol substrates, and purified protein (0.5-1 mg per reaction) was used for the initial screening according to Jing et al, (2019) with some motifications. The reaction mixture was incubated for 30 min at 30°C, the reaction was stopped by adding reaction solution of UDP-Glo™ assay reagent (Sheikh et al, 2017). Three biological replicates were carried out.…”

Section: Enzymatic Activity Assaymentioning

confidence: 99%

Glucosyltransferase CsUGT78A14 Regulates Flavonols Accumulation and Reactive Oxygen Species Scavenging in Response to Cold Stress in Camellia sinensis

et al. 2019

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Glycosyltransferases (UGTs) play diverse roles in cellular metabolism by altering regulatory metabolites activities. However, the physiological roles of most members of UGTs in crops in response to abiotic stresses are unknown. We have identified a novel glycosyltransferase CsUGT78A14 in tea crops, an important economic crops, whose expression is strongly induced by cold stress. Biochemical analyses confirmed that CsUGT78A14-1 showed the highest activity toward kaempferol and is involved in the biosynthesis of kaempferol-diglucoside, whereas the product of CsUGT78A14-2, which differs from CsUGT78A14-1 by a single amino acid, was identified as 3-O-glucoside. The accumulation of kaempferol monoglucosides and diglucosides was consistent with the expression levels of CsUGT78A14 in response to cold stress, as well as in different tissues and genotypes of tea plants. Down-regulation of CsUGT78A14 resulted in reduced accumulation of flavonols, reactive oxygen species (ROS) scavenging capacity and finally reduced tea plant stress tolerance under cold stress. The antioxidant capacity of flavonols aglycon was enhanced by glucosylation catalyzed by CsUGT78A14. The results demonstrate that CsUGT78A14 plays a critical role in cold stress by increasing flavonols accumulation and ROS scavenging capacity, providing novel insights into the biological role of UGTs and flavonoids in plants.

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“…Most glycosyltransferases are capable of specifically hydrolyzing their donor substrates by transferring the sugar to water in the absence of an appropriate acceptor (18). A screen for the ability of purified His 6 -Glt1 to hydrolyze six different UDPsugar donors, based on generation of UDP, revealed strong selectivity for UDP-Glc ( Fig.…”

Section: Activity Of Recombinant Glt1mentioning

confidence: 99%

“…Hydrolysis of UDP-sugars was conducted using the UDP-Glo assay (Promega) as described (18). Briefly, His 6 -Glt1 (after Q-column purification) was incubated in the presence of 50 M sugar nucleotides in 20-l reactions containing 50 mM HEPES-NaOH (pH 7.4), 2 mM MnCl 2 , 5 mM DTT at 37°C for 16 h, and activity was quantitated based on conversion of the UDP reaction product to ATP.…”

Section: Glt1 Enzyme Activity Assaysmentioning

confidence: 99%

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

Rahman¹,

Mandalasi

Zhao

et al. 2017

Journal of Biological Chemistry

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Skp1 is a subunit of the SCF (kp1/ullin 1/-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from is hydroxylated by an O-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba , where it regulates SCF assembly and O-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in , generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [H]glucose from UDP-[H]glucose to the trisaccharide form of Skp1 in a -dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 inΔ extracts, suggesting specificity for Skp1. -knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

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Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases

Cited by 45 publications

References 37 publications

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Glucosyltransferase CsUGT78A14 Regulates Flavonols Accumulation and Reactive Oxygen Species Scavenging in Response to Cold Stress in Camellia sinensis

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

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