Academic literature on the topic 'C-glycosyltransferase'

ABSTRACT Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

ABSTRACT A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in ‘Valencia’ sweet orange ( Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730–874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

ABSTRACT Pseudomonas aeruginosa Pa5196 produces type IV pilins modified with unusual α1,5-linked d-arabinofuranose (α1,5-d-Araf) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from Mycobacterium spp. In this work, we identify a second strain of P. aeruginosa, PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of αβ-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of α1,5-d-Araf were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the Mycobacterium arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196 pilA mutant complemented with other Pseudomonas pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpW's pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.

Glycosyltransferases (GTs), which are distributed widely in various organisms, including bacteria, fungi, plants and animals, play a role in synthesizing biological compounds. Glycosyltransferase-1 fromBacillus cereus(BcGT-1), which is capable of transferring glucose to small molecules such as kaempferol and quercetin, has been identified as a member of the family 1 glycosyltransferases which utilize uridine diphosphate glucose (UDP-glucose) as the sugar donor.BcGT-1 (molecular mass 45.5 kDa) has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction ofBcGT-1 crystals to 2.10 Å resolution, the crystal belonged to space groupP1, with unit-cell parametersa= 54.56,b= 84.81,c= 100.12 Å, α = 78.36, β = 84.66, γ = 84.84°. Preliminary analysis indicates the presence of fourBcGT-1 molecules in the asymmetric unit with a solvent content of 50.27%.

C-Glycosylation presents a rare mode of sugar attachment to the core structure of natural products and is catalyzed by a special type of Leloir C-glycosyltransferases (C-GTs). Elucidation of mechanistic principles for these glycosyltransferases (GTs) is of fundamental interest, and it could also contribute to the development of new biocatalysts for the synthesis of valuable C-glycosides, potentially serving as analogues of the highly hydrolysis-sensitive O‑glycosides. Enzymatic glucosylation of the natural dihydrochalcone phloretin from UDP‑D-glucose was applied as a model reaction in the study of a structurally and functionally homologous pair of plant glucosyltransferases, where the enzyme from rice (Oryza sativa) was specific for C-glycosylation and the enzyme from pear (Pyrus communis) was specific for O-glycosylation. We show that distinct active-site motifs are used by the two enzymes to differentiate between C- and O-glucosylation of the phloretin acceptor. An enzyme design concept is therefore developed where exchange of active-site motifs results in a reversible switch between C/O-glycosyltransferase (C/O-GT) activity. Mechanistic proposal for enzymatic C-glycosylation involves a single nucleophilic displacement at the glucosyl anomeric carbon, proceeding through an oxocarbenium ion-like transition state. Alternatively, the reaction could be described as Friedel–Crafts-like direct alkylation of the phenolic acceptor.

Arabinogalactan proteins (AGPs) are abundant extracellular proteoglycans that are found in most plant species and involved in many cellular processes, such as cell proliferation and survival, pattern formation, and growth, and in plant microbe interaction. AGPs are synthesized by posttranslational O-glycosylation of proteins and attached glycan part often constitutes greater than 90% of the molecule. Subtle altered glycan structures during development have been considered to function as developmental markers on the cell surface, but little is known concerning the molecular mechanisms. My group has been working on glycosylation enzymes (glycosyltransferases) of AGPs to investigate glycan function of the molecule. This review summarizes the recent findings from my group as for AtGalT31A, AtGlcAT14A-C, and AtGalT29A of Arabidopsis thaliana with a specific focus on the (i) biochemical enzyme activities; (ii) subcellular compartments targeted by the glycosyltransferases; and (iii) protein-protein interactions. I also discuss application aspect of glycosyltransferase in improving AGP-based product used in industry, for example, gum arabic.

Bacterial iron acquisition by the means of enterobactin (ENT) is constrained in mammalian hosts due to ENT-binding proteins such as siderocalin and serum albumin. To evade sequestration by these proteins, ENT can be modified by the C glycosyltransferase IroB, which is located in the iroA locus of Salmonella and certain extraintestinal E. coli strains such as uropathogenic E. coli CFT073. The glycosylation of ENT has been reported to be a bacterial evasion mechanism to restore the iron scavenging ability of ENT in the presence of mammalian ENT-binding proteins by the installation of a steric impediment. The C glycosyltransferase IroB catalyses the transfer of a glucose moiety to the DHB subunit of ENT under formation of a C-C bond between the anomeric C1 of the glucose moiety and the C5 of the 2,3-DHB subunit of ENT. The formation of mono-, di- and triglycosylated Ent (MGE/DGE/TGE) products where observed in vitro. The formation of a C-C bond is remarkable because of its chemical stability and resilience against enzymatic degradation. In this M.Sc. thesis, we initially identified the iroB gene product in the iroA harbouring E. coli strain Nissle 1917 on transcriptional and translational level and expressed and purified IroB recombinant. Then, we investigated the mechanism of the C-C bond formation catalysed by IroB in vitro. Based on the hypothesis that deprotonation of the catechol 2 hydroxyl renders the catechol C5 para to the 2-hydroxyl nucleophilic, the C-C bond would then be formed in a general SN2 reaction between the attacking nucleophile and the anomeric carbon of glucose, which is further facilitated by the excellent phosphate leaving group of the UDP-glucose donor. By the means of homology modelling and superposition strategies, we were able to identify the binding sites of the glycosyl donor UDP-glucose and the glycosyl acceptor ENT and to locate residues that could potentially act as base catalysts to increase the phenolate anionic character of the 2,3-DHB subunit during catalysis. We established an activity assay for IroB, separated products arising from IroB activity by reversed phase chromatography and compared so the activity of wild-type IroB and several variants. Additionally, all variants were characterized biophysically, mainly to confirm that the structural integrity was not impaired by mutations. Ultimately, our results enable us to propose a mechanism for C-glycosylation of IroB that is consistent with other glycosyltransferases found in nature.

碩士
國立臺灣大學
分子與細胞生物學研究所
103
Epithelial cells are linked by cell-cell junctions that hold the tissue together, and function as a protective barrier. In the nematode Caenorhabditis elegans, epidermal seam cells are arranged in longitudinal rows on the left and right sides of the body and are embedded in the cylindrical hyp7 syncytium. Seam cells and hyp7 are connected by adhesion junctions along their apical borders. These cell junctions are essential for cell polarity, adhesion and innate immunity. In the 4th larval (L4) stage, seam cells become terminally differentiated and are fused to make one syncytium of 16 cells on each side. Using AJM-1::GFP and HMR-1::GFP fusion proteins that mark the apical adhesion junction, I observed two almost parallel lines that run along the seam syncytium boundary on each side of the body in the wild-type adult animals. However, in mutants defective in blmp-1, which encodes a zinc finger transcription factor similar to mammalian transcriptional repressor BLIMP-1(B lymphocyte-induced maturation protein 1), the apical epithelial junctions showed a bubble-like arrangement in seam cells in adult. By performing the time-course analysis of the AJM-1::GFP pattern in hypodermis, I found that, in the blmp-1 mutant, seam cell fusion and the adhesion junction arrangement is normal in L4, but the border between seam cell and hyp7 became irregular and the opposite sides of the border started became attached at multiple points during the L4/adult molt. The epidermal basolateral region marker LET-413::GFP showed no detectable abnormality in the basal region of the seam syncytium. In addition, inactivation of blmp-1 by RNA interference in either seam or hyp7 resulted in bubble-like apical epithelial junctions, showing that blmp-1 is essential in both seam and hyp7 to maintain the normal apical surface of the seam syncitium. Since BLMP-1 can function as a transcriptional repressor, this seam cell apical surface defect may be caused by abnormally high expression of some target genes. Using candidate genes approach and a function test, I found that bus-8 RNAi significantly reduced the percentage of blmp-1 mutants with the abnormal apical seam cell shape. BUS-8 is predicted as a mannosyltransferases involved in protein glycosylation, such as bus-2, bus-4, bus-12, partially rescued the apical seam cell shape defect of the blmp-1 mutant. On the basis of these data, I proposed that loss of blmp-1 caused the abnormality of apical seam cell shape due to abnormal protein glycosylation by BUS-8, in particular, and BUS-2, BUS-4 and BUS-12, in part, in seam and/ or hyp7 cells and that proper glycosylation of extracellular proteins or membrane proteins is important for the maintenance of the apical epithelial cell shape.

Colour is an important determinant of quality and customer appeal for Asian noodles that are made from bread wheat (Triticum aestivum L.). The Asian noodle market represents approximately one third of wheat exports from Australia and as a consequence maintaining and improving colour for noodles is an important research and breeding objective. The focus of this project is yellow alkaline noodles (YAN) prepared using wheat flour and alkaline salts, sodium and potassium carbonate, and for which a bright yellow colour is desired. Xanthophylls, primarily lutein, and apigenin di-Cglycosides (ACGs) have been shown to be important components of this yellow colour. ACGs were of particular interest since, in contrast to lutein, the content in flour could be increased without adverse effects on colour of other end-products. There was little information either on the genetic variation for ACG content or the mechanism and genetic control of biosynthesis which was surprising in view of their putative role in a wide range of plant processes, food colour and flavour, and possibly human health. The aims of this project were to provide new information on the role of ACGs in YAN colour and genetic regulation of their biosynthesis. To achieve this aims: genetic variation in grain ACG traits in bread wheat and related species was surveyed, the quantitative contribution of ACG to the yellow colour of YAN was determined and compared to lutein, QTL for ACG content and composition were located, and candidate genes associated with variation in ACG composition identified. Substantial variation in both grain ACG content and the ratio, ACG1/ACG2, were identified within bread wheat cultivars and related species. Genotype controlled the major portion of the variation. ACG content appeared to be a multigenic trait whereas variation in ACG1/ACG2 was associated with a limited number of chromosomes, in particular chromosomes 1B, 7B and 7D. In the absence of chromosome 7B (Chinese Spring 7B nullisomics) there was a substantial increase in ACG1/ACG2, i.e. a relative increase in the glucose-containing isomer, possibly indicating the presence of a Cglycosyltransferase on 7B with specificity for UDP-galactose. A similar phenotype observed in some wheat cultivars could be explained by a deletion or mutation of a gene controlling this enzyme. The results suggest that it should be possible to manipulate both ACG content and composition through breeding. Only 30% of ACG (means 19.3 µg/g) is recovered in flour, which contributed to 1 to 3 CIE b* units to the part of the yellow colour of yellow alkaline noodles (YAN) that develops specifically in the presence of alkali. The relatively low recovery of ACG in flour contrasts with the high recovery of lutein (90%, with means 1.011 µg/g). Since the difference between white salted noodles (WSN) and YAN is approximately 6 b* units, this would indicate that another unidentified compound(s) is responsible for the difference. Potential for ACG0-based improvement of bread wheat cultivars for YAN yellowness is likely to be limited by the range of genetic variation, the location of ACG in grain tissues that are largely discarded during milling and the lack of correlation between grain and flour ACG content. Moreover, the observed variation in ACG recovery in small scale milling was not reflected in larger scale milling anticipated to better represent commercial practice. The improvement in flour recovery and the amount of ACG recovered in the flour were not significant and not enough to achieve the yellowness of commercial noodles. Selection that requires larger scale milling is costly, time consuming and not applicable to early generation screening. In this context, further work on QTL associated with variation in ACG content and development of marker-assisted-selection would be very useful. Addition of thirteen new markers to the QTL region for ACG trait on chromosome 7BS in a Sunco/Tasman doubled haploid population reduced the size of the QTL interval from 28.8cM to approximately 5.5cM. In this revised 7BS map, the major QTL for ACG1 and ACG2 content as well as ACG1/ACG2 ratio were detected within 4.7cM of SSR marker Xwmc76. The QTL region linked to Xwmc76 was shown to be syntenic with a region in rice chromosome 6S between AP005387 and AP005761 and a region on Brachypodium chromosome 1. Based on these comparisons, the most likely candidate gene associated with variation in ACG composition appeared to be a glycosyltransferase. Alternate alleles at the 7BS QTL may be associated with amino acid changes within the C glycosyltransferase that shift the substrate specificity from galactose (ACG2, Tasman) to glucose (ACG1, Sunco). Alternatively, based on a comparison of Chinese Spring nullisomic-tetrasomic lines where nullisomic 7B was associated with a phenotype similar to Sunco, it is possible that Sunco contains a null allele. Other candidate genes located on the same chromosome that could potentially be involved in ACG biosynthesis were identified and included a sugar transporter, which could determine the relative sizes of the available pools of UDP-glucose and UDPgalactose, an epimerase required for inter-conversion of these sugars, other glycosyltransferases and a flavone-2-hydroxylase (F2H) involved in the first committed step in the pathway to ACG. Research approaches that could be used to validate the role of the candidate gene are discussed along with other options for improving the colour of wheat cultivars for the YAN market. Options for utilizing ACG as yellow pigment of noodles might include incorporating the embryo or seed coat materials (pollard and bran) into the flour after milling and genetic modification of bread wheat to achieve ACG expression in the starchy endosperm.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012

Glycosyl group transfer underlies the biosynthesis and breakdown of all nucleotides, polysaccharides, glycoproteins, glycolipids, and glycosylated nucleic acids, as well as certain DNA repair processes. Glycosyl transfer consists of the transfer of the anomeric carbon of a sugar derivative from one acceptor to another, as in, which describes the transfer of a generic pyranosyl ring between nucleophilic atoms :X and :Y of acceptor molecules. The stereochemistry at the anomeric carbon is not specified in eq. 12-1, but the leaving group occupies the axial position in an α-anomer or the equatorial position in a β-anomer. The overall transfer can proceed with either retention or inversion of configuration. In biochemistry, the acceptor atoms can be oxygen, nitrogen, sulfur, or in the biosynthesis of C-nucleosides even carbon. The great majority of biological glycosyl transfer reactions involve transfer between oxygen atoms of different acceptor molecules. Enzymes catalyzing glycosyl transfer are broadly grouped according to whether the acceptor :Y–R2 in is water or another molecule. In the actions of glycosidases, the acceptor is water, and glycosyl transfer results in hydrolysis of a glycoside, a practically irreversible process in dilute aqueous solutions. In the action of glycosyltransferases, the acceptors are molecules with hydroxyl, amide, amine, sulfhydryl, or phosphate groups. The simplest nonenzymatic glycosyl transfer reaction is the hydrolysis of a glycoside, and early studies revealed the fundamental fact that glycosides are much less reactive toward hydrolysis in basic solutions than in acidic solutions. This fact underlies much that is known about the mechanism of glycosyl transfer; that is, the anomeric carbon of a glycoside is remarkably unreactive toward direct nucleophilic attack, but it becomes reactive when one of the oxygens is protonated by an acid, as illustrated in fig. 12-1 for the acid-catalyzed hydrolysis of a generic glycoside. The reaction by both mechanisms in fig. 12-1 proceeds by pre-equilibrium protonation of the glycoside to form oxonium ion intermediates, which are subject to hydrolysis by water. The two mechanisms in fig. 12-1 are of interest. The mechanism proceeding through exocyclic cleavage of the glycoside has historically been regarded as the more likely, and for this reason, the route through endocyclic cleavage has received little consideration.