UDP-glucosyltransferase activity of housefly microsomal fraction.
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ABSTRACT:
SUBMITTER: Morello A
PROVIDER: S-EPMC1186444 | biostudies-other | 1979 Mar
REPOSITORIES: biostudies-other
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Project description:The monoterpene linalool is a major contributor to aroma and flavor in peach (Prunus persica) fruit. It accumulates during fruit ripening, where up to ~40% of the compound is present in a non-volatile glycosylated form, which affects flavor quality and consumer perception by retronasal perception during tasting. Despite the importance of this sequestration to flavor, the UDP-glycosyltransferase (UGT) responsible for linalool glycosylation has not been identified in peach. UGT gene expression during peach fruit ripening and among different peach cultivars was analyzed using RNA sequencing, and transcripts correlated with linalyl-?-d-glucoside were selected as candidates for functional analysis. Kinetic resolution of a racemic mixture of R,S-linalool was shown for PpUGT85A2, with a slight preference for S-(+)-linalool. PpUGT85A2 was shown to catalyze synthesis of linalyl-?-d-glucoside in vitro, although it did not exhibit the highest enzyme activity between tested substrates. Subcellular localization of PpUGT85A2 in the cytoplasm and nucleus was detected. Application of linalool to peach leaf disks promoted PpUGT85A2 expression and linalyl-?-d-glucoside generation. Transient expression in peach fruit and stable overexpression in tobacco and Arabidopsis resulted in significant accumulation of linalyl-?-d-glucoside in vivo. Taken together, the results indicate that PpUGT85A2 expression is a major control point predicting linalyl-?-d-glucoside content.
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Project description:BACKGROUND: Glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. A class of UDP-glycosyltransferases (UGTs) is involved in this process. Insect UGTs play important roles in several processes, including detoxication of substrates such as plant allelochemicals, cuticle formation, pigmentation, and olfaction. Identification and characterization of Bombyx mori UGT genes could provide valuable basic information for this important family and explain the detoxication mechanism and other processes in insects. RESULTS: Taking advantage of the newly assembled genome sequence, we performed a genome-wide analysis of the candidate UGT family in the silkworm, B. mori. Based on UGT signature and their similarity to UGT homologs from other organisms, we identified 42 putative silkworm UGT genes. Most of them are clustered on the silkworm chromosomes, with two major clusters on chromosomes 7 and 28, respectively. The phylogenetic analysis of these identified 42 UGT protein sequences revealed five major groups. A comparison of the silkworm UGTs with homologs from other sequenced insect genomes indicated that some UGTs are silkworm-specific genes. The expression patterns of these candidate genes were investigated with known expressed sequence tags (ESTs), microarray data, and RT-PCR method. In total, 36 genes were expressed in tissues examined and showed different patterns of expression profile, indicating that these UGT genes might have different functions. CONCLUSION: B. mori possesses a largest insect UGT gene family characterized to date, including 42 genes. Phylogenetic analysis, genomic organization and expression profiles provide an overview for the silkworm UGTs and facilitate their functional studies in future.
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Project description:Flowers of the butterfly pea (Clitoria ternatea) accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in C. ternatea by UDP-glucose:anthocyanidin 3-O-glucosyltransferase (Ct3GT-A; AB185904). To elucidate the structure-function relationship of Ct3GT-A, recombinant Ct3GT-A was expressed in Escherichia coli and its tertiary structure was determined to 1.85 Å resolution by using X-ray crystallography. The structure of Ct3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like ?/?/? domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of Ct3GT-A with that of the flavonoid glycosyltransferase VvGT1 from red grape (Vitis vinifera) in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in Ct3GT-A, but certain residues of VvGT1 involved in binding kaempferol were found to be substituted in Ct3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.
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Project description:Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP-glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP-glucose to N-linked Man9-GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.
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Project description:Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.
| S-EPMC3756913 | biostudies-literature
Project description:The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.
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Project description:Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-?-d-glucopyranosyl-6-O-[?-l-rhamnopyrano-(1?2)-?-d-glucopyranosyl]-dammar-24-ene-3?,6?,12?,20S-tetraol (1), 3,20-di-O-?-d-glucopyranosyl-6-O-[?-d-xylopyranosyl-(1?2)-?-d-glucopyranosyl]-dammar-24-ene-3?,6?,12?,20S-tetraol (6), 3-O-?-d-glucopyranosyl-6-O-[?-d-glucopyranosyl-(1?2)-?-d-glucopyranosyl]-dammar-24-ene-3?,6?,12?,20S-tetraol (3), 3,12-di-O-?-d-glucopyranosyl-6-O-[?-d-glucopyranosyl-(1?2)-?-d-glucopyranosyl]-dammar-24-ene-3?,6?,12?,20S-tetraol (2), 3,6-di-O-?-d-glucopyranosyl-dammar-24-ene-3?,6?,12?,20S-tetraol (5), and 3,6,12-tri-O-?-d-glucopyranosyl-dammar-24-ene-3?,6?,12?,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0?10.0. The activity of UGT109A1 was considerably enhanced by Mg2+, Mn2+, and Ca2+, but was obviously reduced by Cu2+, Co2+, and Zn2+. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.
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Project description:Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.
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Project description:Ginkgo biloba L., a "living fossil" and medicinal plant, is a well-known rich source of bioactive flavonoids. The molecular mechanism underlying the biosynthesis of flavonoid glucosides, the predominant flavonoids in G. biloba, remains unclear. To better understand flavonoid glucosylation in G. biloba, we generated a transcriptomic dataset of G. biloba leaf tissue by high-throughput RNA sequencing. We identified 25 putative UDP-glycosyltransferase (UGT) unigenes that are potentially involved in the flavonoid glycosylation. Among them, we successfully isolated and expressed eight UGT genes in Escherichia coli, and found that recombinant UGT716A1 protein was active toward broad range of flavonoid/phenylpropanoid substrates. In particular, we discovered the first recombinant UGT protein, UGT716A1 from G. biloba, possessing unique activity toward flavanol gallates that have been extensively documented to have significant bioactivity relating to human health. UGT716A1 expression level paralleled the flavonoid distribution pattern in G. biloba. Ectopic over-expression of UGT716A1 in Arabidopsis thaliana led to increased accumulation of several flavonol glucosides. Identification and comparison of the in vitro enzymatic activity of UGT716A1 homologs revealed a UGT from the primitive land species Physcomitrella patens also showed broader substrate spectrum than those from higher plants A. thaliana, Vitis vinifera, and Medicago truncatula. The characterization of UGT716A1 from G. biloba bridges a gap in the evolutionary history of UGTs in gymnosperms. We also discuss the implication of UGT716A1 for biosynthesis, evolution, and bioengineering of diverse glucosylated flavonoids.
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Project description:Clostridium difficile is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. The organism produces two homologous toxins, TcdA and TcdB, which enter and disrupt host cell function by glucosylating and thereby inactivating key signalling molecules within the host. As a toxin-mediated disease, there has been a significant interest in identifying small molecule inhibitors of the toxins' glucosyltransferase activities. This study was initiated as part of an effort to identify the mode of inhibition for a small molecule inhibitor of glucosyltransferase activity called apigenin. In the course of trying to get co-crystals with this inhibitor, we determined five different structures of the TcdA and TcdB glucosyltransferase domains and made use of a non-hydrolyzable UDP-glucose substrate. While we were able to visualize apigenin bound in one of our structures, the site was a crystal packing interface and not likely to explain the mode of inhibition. Nevertheless, the structure allowed us to capture an apo-state (one without the sugar nucleotide substrate) of the TcdB glycosyltransferase domain that had not been previously observed. Comparison of this structure with structures obtained in the presence of a non-hydrolyzable UDP-glucose analogue have allowed us to document multiple conformations of a C-terminal loop important for catalysis. We present our analysis of these five new structures with the hope that it will advance inhibitor design efforts for this important class of biological toxins.
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