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Query: UNIPROT:P06889 (Mol)
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T4 phage beta-glucosyltransferase (BGT) modifies T4 DNA. We crystallized BGT with UDP-glucose and a 13mer DNA fragment containing an abasic site. We obtained two crystal structures of a ternary complex BGT-UDP-DNA at 1.8A and 2.5A resolution, one with a Tris molecule and the other with a metal ion at the active site. Both structures reveal a large distortion in the bound DNA. BGT flips the deoxyribose moiety at the abasic site to an extra-helical position and induces a 40 degrees bend in the DNA with a marked widening of the major groove. The Tris molecule mimics the glucose moiety in its transition state. The base-flipping mechanism, which has so far been observed only for glycosylases, methyltransferases and endonucleases, is now reported for a glucosyltransferase. BGT is unique in binding and inserting a loop into the DNA duplex through the major groove only. Furthermore, BGT compresses the backbone DNA one base further than the target base on the 3'-side.
J Mol Biol 2002 Nov 29
PMID:A base-flipping mechanism for the T4 phage beta-glucosyltransferase and identification of a transition-state analog. 1244 83

The waa locus on the chromosome of Salmonella enterica encodes enzymes involved in the assembly of the core oligosaccharide region of the lipopolysaccharide (LPS) molecule. To date, there are two known core structures in Salmonella, represented by serovars Typhimurium (subspecies I) and Arizonae (subspecies IIIA). The waa locus for serovar Typhimurium has been characterized. Here, the corresponding locus from serovar Arizonae is described, and the molecular basis for the distinctive structures is established. Eleven of the 13 open reading frames (ORFs) are shared by the two loci and encode conserved proteins of known function. Two polymorphic regions distinguish the waa loci. One involves the waaK gene, the product of which adds a terminal alpha-1,2-linked N-acetylglucosamine residue that characterizes the serovar Typhimurium core oligosaccharide. There is an extensive internal deletion within waaK of serovar Arizonae. The serovar Arizonae locus contains a novel ORF (waaH) between the waaB and waaP genes. Structural analyses and in vitro glycosyltransferase assays identified WaaH as the UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase responsible for the addition of the characteristic terminal glucose residue found in serovar Arizonae. Isolates comprising the Salmonella Reference Collections, SARC (representing the eight subspecies of S. enterica) and SARB (representing subspecies I), were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies identified additional waa locus polymorphisms, shedding light on the genetic basis for diversity in the LPS core oligosaccharides of Salmonella isolates and identifying potential sources of further novel LPS structures.
Mol Microbiol 2002 Dec
PMID:Molecular diversity of the genetic loci responsible for lipopolysaccharide core oligosaccharide assembly within the genus Salmonella. 1245 17

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.
Mol Genet Genomics 2003 Mar
PMID:Characterization of the ugpG gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer Sphingomonas paucimobilis ATCC 31461. 1265 8

The ability to regulate energy balance at both the cellular and whole body level is an essential process of life. As western society has shifted to a higher caloric diet and more sedentary lifestyle, the incidence of type 2 diabetes (non-insulin-dependent diabetes mellitus) has increased to epidemic proportions. Thus, type 2 diabetes has been described as a disease of 'chronic overnutrition'. There are abundant data to support the relationship between nutrient availability and insulin action. However, there have been multiple hypotheses and debates as to the mechanism by which nutrient availability modulates insulin signaling and how excess nutrients lead to insulin resistance. One well-established pathway for nutrient sensing is the hexosamine biosynthetic pathway (HSP), which produces the acetylated aminosugar nucleotide uridine 5'-diphospho-N-acetylglucosamine (UDP-Glc-NAc) as its end product. Since UDP-GlcNAc is the donor substrate for modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (O-GlcNAc), the possibility of this posttranslational modification serving as the nutrient sensor has been proposed. We have recently directly tested this model in adipocytes by examining the effect of elevated levels of O-GlcNAc on insulin-stimulated glucose uptake. In this review, we summarize the existing work that implicates the HSP and O-GlcNAc modification as nutrient sensors and regulators of insulin signaling.
Cell Mol Life Sci 2003 Feb
PMID:A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance. 1267 87

Identification of a G protein-coupled receptor activated by UDP-glucose led us to develop a sensitive and specific assay for UDP-glucose mass and to test whether this sugar nucleotide is released as an extracellular signaling molecule. Mechanical stimulation of 1321N1 human astrocytoma cells by a change of medium resulted in an increase in extracellular levels of both ATP and UDP-glucose. Whereas ATP levels peaked within 10 min and subsequently returned to resting extracellular levels of 3 nM, UDP-glucose levels attained a steady state that exceeded that of resting ATP levels by 3- to 5-fold for at least 3 h. Similar rates of basal release of UDP-glucose and ATP (72 and 81 fmol/min/10(6) cells) combined with a rate of UDP-glucose metabolism approximately three times lower than ATP hydrolysis account for the elevated extracellular UDP-glucose levels on resting cells. A medium change also resulted in rapid appearance of UDP-glucose on the luminal surface of highly differentiated polarized human airway epithelial cells but at levels 2- to 3-fold lower than ATP. However, nucleotide sugar levels increased 3- to 5-fold over the ensuing 2 h, whereas ATP levels decayed to a resting level; consequently, resting extracellular UDP-glucose levels exceeded those of ATP by 5- to 10-fold. UDP-glucose also was observed at levels that equaled or exceeded those of ATP in the extracellular medium of Calu-3 airway epithelial, COS-7, CHO-K1, and C6 glioma cells. Consistent with the observation of significant extracellular UDP-glucose levels, expression of the UDP-glucose-activated P2Y(14) receptor in COS-7 cells resulted in G protein-promoted inositol phosphate accumulation that was partially reversed by enzymatic removal of UDP-glucose from the medium. Taken together, these results indicate constitutive release of UDP-glucose from physiologically relevant tissues and suggest that UDP-glucose acts as an autocrine activator of the P2Y(14) receptor. Because cellular UDP-glucose is concentrated in the lumen of the endoplasmic reticulum, we speculate that UDP-glucose release may occur as a result of vesicle transport during trafficking of glycoproteins to the plasma membrane.
Mol Pharmacol 2003 May
PMID:Release of cellular UDP-glucose as a potential extracellular signaling molecule. 1269 47

Over a broad taxonomic range that spans monocots and dicots, upstream enzymes of the anthocyanin pigment pathway have evolved less rapidly than downstream enzymes. In this article we show that this pattern is also evident within the genus Ipomoea. Specifically, the most upstream enzyme, chalcone synthase (CHS-D), evolves more slowly than the two most downstream enzymes, ancyocyanidin synthase (ANS) and UDP glucose flavonoid 3-oxy-glucosyltransferase (UFGT). This pattern appears not to be due to variation in mutation rates, because the CHS-D gene exhibits higher synonymous substitution rates than the genes for the other two enzymes. Codon-based tests for positive selection suggest that it has been negligible or absent in all three genes. In addition, the mean number of indel-creating events is four times as high in the downstream genes as in CHS-D. Unlike the downstream genes, CHS-D also exhibits evidence of codon bias. Together, the evidence suggests that the difference in nonsynonymous substitution rates between upstream and downstream genes is due to relaxed constraint on the downstream genes rather than a greater frequency of positively selected substitutions.
Mol Biol Evol 2003 Nov
PMID:Evolutionary rate variation in anthocyanin pathway genes. 1288 63

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.
Mol Biol Cell 2003 Sep
PMID:The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum. 1297 44

Many plant autocatalytic glycosyltransferases are implicated in plant polysaccharide biosynthesis. Cloning of cDNAs encoding potato (Solanum tuberosum L.) UDP-Glc:protein transglucosylase (UPTG, EC 2.4.1.112) and expression of the cDNA clone E11 in Escherichia coli have been previously reported. Here, we studied the functional expression of a second cDNA of the enzyme (E2 clone). Northern blots analysis, with specific cDNA probes for the two UPTG isoforms, showed a differential expression pattern of mRNA levels in different potato tissues. Moreover, both UPTG recombinant enzymes showed different kinetic parameters. The recombinant protein encoded by E2 clone has an apparent Imax for UDP-Xyl and UDP-Gal, significantly higher than for UDP-Glc. The Km values for UDP-Glc were 0.45-0.71 microM and the values for UDP-Xyl and UDP-Gal were slightly higher than that of the UDP-Glc (1.2-2.71 microM) for both UPTG recombinant enzymes. The present study revealed further evidence for the proposed role of UPTG in the synthesis of cell wall polysaccharide. It was found a correlation between UPTG transcript levels and the growing state of the tissues in which there was an active synthesis of cell wall components. Southern blot analysis indicates that at least three genes encoding UPTG are present in potato genome. Phylogenetic analysis of both UPTG recombinant proteins showed that they are members of the RGP subfamilies from dicots.
Plant Mol Biol 2003 Jul
PMID:Characterization of UDP-glucose:protein transglucosylase genes from potato. 1367 61

We have recently shown that UDP-glucose, and some related UDP-sugars, are potent agonists of the novel G protein-coupled receptor GPR105 (recently re-named P2Y(14)). GPR105 is widely expressed throughout many brain regions and peripheral tissues of human and rodents, and couples to a pertussis toxin-sensitive G protein. To further characterise the role of GPR105, we demonstrate by immunohistochemistry with receptor-specific antiserum that GPR105 protein is widely distributed throughout the post mortem human brain where it is localised to glial cells, and specifically co-localises with astrocytes. Using quantitative RT-PCR we also show that GPR105 mRNA exhibits a restricted expression profile in an array of human cell lines and primary cells, with prominent expression detected in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. To investigate the G protein selectivity of GPR105, we used chimeric Galpha subunits (Galpha(qi5), Galpha(qo5), and Galpha(qs5)) and an intracellular Ca(2+) mobilisation assay to demonstrate that GPR105 couples to Galpha subunits of the G(i/o) family but not to G(s) family proteins or to endogenous G(q/11) proteins in HEK-293 cells. Finally, we show that expression of GPR105 mRNA in the rat brain is up-regulated by immunologic challenge with lipopolysaccharide. Based on these observations, we propose that G(i/o)-coupled GPR105 might play an important role in peripheral and neuroimmune function in response to extracellular UDP-sugars.
Brain Res Mol Brain Res 2003 Oct 21
PMID:GPR105, a novel Gi/o-coupled UDP-glucose receptor expressed on brain glia and peripheral immune cells, is regulated by immunologic challenge: possible role in neuroimmune function. 1455 50

Trypanosoma cruzi incorporates galactose into many of its cell-surface glycoconjugates but it is unable to transport this sugar through its hexose transporter. Epimerisation of UDP-glucose to UDP-galactose by UDP-glucose 4'-epimerase may be the only way that the parasites can obtain galactose. Here, we describe cloning the T. cruzi UDP-Glc 4'-epimerase (TcGALE) gene and show that it is functional by complementing an Escherichia coli epimerase-deficient strain. The T. cruzi GALE gene encodes a 42.4 kDa protein and the recombinant protein expressed in E. coli is a homodimer in solution with a specific activity of 3.8 U mg(-1) and K(m) for UDP-Gal of 114 microM. Unlike the human epimerase, T. cruzi UDP-Glc 4'-epimerase is unable to inter-convert UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. This may explain why T. cruzi initiates O-glycosylation of its abundant GPI-anchored surface mucins via GlcNAcalpha1-O-Thr/Ser rather than the GalNAcalpha1-O-Thr/Ser linkage that is common for mucins from many other eukaryotes.
Mol Biochem Parasitol 2003 Nov
PMID:Cloning and characterisation of the UDP-glucose 4'-epimerase of Trypanosoma cruzi. 1456 36


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