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1
Dumler, F., and P. Cortes. "Uracil ribonucleotide metabolism in rat and human glomerular epithelial and mesangial cells." American Journal of Physiology-Cell Physiology 255, no. 6 (December 1, 1988): C712—C718. http://dx.doi.org/10.1152/ajpcell.1988.255.6.c712.
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Uridine diphosphosugars (UDP-sugars: UDP-N-acetylglucosamine, UDP-glucose, and UDP-glucuronic acid) are essential coenzymes for the synthesis of glomerular basement membrane and mesangial matrix (GBM-MM). This study has characterized UDP-sugar metabolism in rat and human glomerular cells in tissue culture. Culture of rat mesangial cells in medium containing dialyzed fetal calf serum resulted in UTP loss (28 +/- 4 nmol.mg DNA-1.h-1); the addition of 2 microM orotate to this medium resulted in net UTP accretion (5.42 +/- 0.06 nmol.mg DNA-1.h-1). Rat mesangial cells demonstrated 16- and 29- to 46-fold greater UTP and UDP-sugar pools, respectively, than whole glomeruli. In human mesangial cells, 6-azauridine (500 microM) decreased UDP-sugar pools by 48% (P less than 0.05), whereas uridine (50 microM) produced a 2.5-fold increase. Human and rat mesangial cells had greater (1.8- to 6.1-fold) UDP-sugar pools than epithelial cells and 1.7-3.4 times greater labeled precursor incorporation into UDP-sugars. In conclusion, glomerular cells utilize both exogenous orotate and uridine for ribonucleotide synthesis, and the extracellular concentration of these precursors markedly influence the formation and cellular content of UDP-sugars. Prominent differences exist between separate glomerular cell populations in their metabolism of UDP-sugars. This may represent diverse activity of glycosylating reactions.
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Sener, Keriman, Zuojun Shen, David S. Newburg, and Edward L. Jarroll. "Amino sugar phosphate levels in Giardia change during cyst wall formation." Microbiology 150, no. 5 (May 1, 2004): 1225–30. http://dx.doi.org/10.1099/mic.0.26898-0.
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The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique β(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.
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Sweeney, C., D. Mackintosh, and R. M. Mason. "UDP-sugar metabolism in Swarm rat chondrosarcoma chondrocytes." Biochemical Journal 290, no. 2 (March 1, 1993): 563–70. http://dx.doi.org/10.1042/bj2900563.
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UDP-sugars and adenine nucleotides were extracted from freshly isolated chondrocytes and primary cell cultures and analysed by anion-exchange h.p.l.c. The pool sizes of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-glucose-galactose, UDP-glucuronate and UDP-xylose were 2.9, 1.2, 2.5, 0.6 and 0.03 nmol/10(6) freshly isolated chondrocytes. When chondrocytes were maintained in Dulbecco's modified Eagle medium supplemented with 15% foetal-bovine serum, synthesis of [35S]proteoglycan and [3H]protein decreased over the first 48 h in culture, as did the pools of UDP-glucuronate and ATP. In contrast, the size of the UDP-N-acetylhexosamine pools underwent little change during culture. [35S]Proteoglycan and [3H]protein syntheses were stimulated in cultures supplemented with serum or insulin compared with those maintained in medium alone, in agreement with previous results. However, the UDP-sugar pool sizes were the same in both supplemented and non-supplemented cultures. In cultures maintained in the presence of [1-3H]glucose, the UDP-sugars were labelled to a constant 3H specific radioactivity which was very similar to that of the labelling medium. UDP-N-acetylhexosamines were labelled to constant 3H specific radioactivity with [6-3H]glucosamine as a precursor, but only about 1 in 375 of these UDP-sugars was derived from the amino sugar in the presence of glucose. The half-life (t1/2) for UDP-hexoses, UDP-glucuronate and UDP-N-acetylhexosamines was about 12, 12 and 50 min respectively.
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Zhu, Xiangming, Florian Stolz, and Richard R. Schmidt. "Synthesis of Thioglycoside-Based UDP-Sugar Analogues." Journal of Organic Chemistry 69, no. 21 (October 2004): 7367–70. http://dx.doi.org/10.1021/jo049077m.
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McDowell, W., G. Weckbecker, D. O. R. Keppler, and R. T. Schwarz. "UDP-glucosamine as a substrate for dolichyl monophosphate glucosamine synthesis." Biochemical Journal 233, no. 3 (February 1, 1986): 749–54. http://dx.doi.org/10.1042/bj2330749.
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The sugar nucleotide analogue UDP-glucosamine was found to function as a sugar donor in microsomal preparations of both chick-embryo cells and rat liver, yielding dolichyl monophosphate glucosamine (Dol-P-GlcN). This was characterized by t.l.c. and retention by DEAE-cellulose. Glucosamine was the only water-soluble product released on mild acid hydrolysis. Dol-P-GlcN did not serve as substrate by transferring its glucosamine moiety to dolichol-linked oligosaccharide. Competition experiments between UDP-[3H]glucose and UDP-glucosamine showed Dol-P-[3H]glucose synthesis to be depressed by 56 or 73% in microsomes from chick-embryo cells and rat liver respectively. The concentrations of the UDP-sugars in this experiment were comparable with those occurring in galactosamine-metabolizing liver. These findings suggest that Dol-P-GlcN, formed as a metabolite of D-galactosamine, may interfere with Dol-P-dependent reactions.
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Cortes, P., F. Dumler, D. L. Paielli, and N. W. Levin. "Glomerular uracil nucleotide synthesis: effects of diabetes and protein intake." American Journal of Physiology-Renal Physiology 255, no. 4 (October 1, 1988): F647—F655. http://dx.doi.org/10.1152/ajprenal.1988.255.4.f647.
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The biosynthesis of uridine 5'-triphosphate (UTP), uridine 5'-diphosphohexoses, and 5'-diphosphohexosamines (UDP-sugars) was studied in isolated rat glomeruli 48 h after streptozotocin-induced diabetes. Compared with control, diabetic glomeruli demonstrated an increase in the following: exogenous orotate utilization, orotate incorporation into UTP and UDP-sugars, UTP accretion rate, and UDP-sugar pool size. Since these phenomena were not associated with enhanced biosynthesis of orotate de novo, the increased glomerular UDP-sugar bioavailability in diabetes is due to enhanced utilization of exogenous orotate. Plasma concentrations of orotate and uridine were measured in control, sham operated, and unilaterally nephrectomized rats receiving 5, 20, or 60% protein diets. The concentration of pyrimidine precursors correlated directly with protein intake, with doubling at the 60% dietary protein level. In conclusion, glomerular uracil ribonucleotide biosynthesis may be modulated by the quantity of dietary protein. Because UDP-sugars are necessary for basement membrane material formation, an increase in their bioavailability may be part of the metabolic change responsible for diabetic glomerulosclerosis. Diets with high protein content could augment this metabolic alteration.
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Oikari, Sanna, Tiia Kettunen, Satu Tiainen, Jukka Häyrinen, Amro Masarwah, Mazen Sudah, Anna Sutela, Ritva Vanninen, Markku Tammi, and Päivi Auvinen. "UDP-sugar accumulation drives hyaluronan synthesis in breast cancer." Matrix Biology 67 (April 2018): 63–74. http://dx.doi.org/10.1016/j.matbio.2017.12.015.
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DO, Su-Il, Ki-Young LEE, and Hee-Nam KIM. "Novel induction of α-lactalbumin-mediated lacdiNAc-R expression in vivo." Biochemical Journal 348, no. 1 (May 9, 2000): 229–34. http://dx.doi.org/10.1042/bj3480229.
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α-Lactalbumin (α-LA) is a regulatory protein by which the mammalian β1,4-galactosyltransferase (β1,4-galT) is induced to utilize glucose as an acceptor instead of N-acetylglucosamine (GlcNAc) during lactose synthesis in mammary gland. α-LA can also modulate β1,4-galT to utilize UDP-N-acetylgalactosamine (UDP-GalNAc) as a donor towards GlcNAc acceptor substrate with high efficiency in vitro [Do, Do and Cummings (1995) J. Biol. Chem. 270, 18447-18451]. In the present study we transfected cDNA encoding bovine α-LA into Lec8 cells and examined whether nucleotide sugar switching of UDP-galactose (UDP-Gal) into UDP-GalNAc occurred in vivo and whether the neo-glycosylation of GalNAcβ1,4GlcNAc-R structure was synthesized in α-LA-stable transfectants. Our studies demonstrate that the stable expression of α-LA in Lec8 cells induces the formation of GalNAcβ1,4GlcNAc-R in vivo through the nucleotide sugar switching of β1,4-galT.
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Ysart, G. E., and R. M. Mason. "Serum factors, growth factors and UDP-sugar metabolism in bovine articular cartilage chondrocytes." Biochemical Journal 303, no. 3 (November 1, 1994): 713–21. http://dx.doi.org/10.1042/bj3030713.
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1. The effect of different batches of fetal bovine serum and of growth factors on [35S]sulphate incorporation into glycosaminoglycans and on UDP-sugar pools in explant cultures of bovine articular cartilage was investigated. 2. [35S]Sulphate incorporation was variably stimulated between 1.2- and 3.5-fold by four different batches of serum. The UDP-glucuronate pool size expanded 4.3-6.5-fold in the presence of serum, even in those cultures in which little stimulation of [35S]sulphate incorporation occurred. The UDP-N-acetylhexosamine and UDP-hexose pools expanded by about 1.5- and 2.0-fold respectively in the presence of serum. UDP-xylose was not detected. 3. Equilibrium-labelling and pulse-chase experiments with D-[1-3H]glucose indicated that the rate of flux through the UDP-sugar pools was unaffected by serum. UDP-hexose, UDP-N-acetylhexosamine and UDP-glucuronate have approximate half-lives (t1/2) of 7, 12 and 3-4 min respectively. At equilibrium, the 3H specific activities of UDP-hexose and UDP-N-acetylhexosamine were very similar but that for the UDP-glucuronate pool was much higher, especially in serum-supplemented cultures. The results suggest that UDP-glucuronate synthesis occurs via a pathway which is independent of the main UDP-hexose pathway. 4. Supplementing cultures with heat-treated serum had no effect on the serum-induced expansion of UDP-sugar pools but stimulation of [35S]sulphate incorporation into glycosaminoglycans was 50% lower than for native serum. Acid-treated serum promoted a 2-fold expansion of the UDP-glucuronate and UDP-N-acetylhexosamine pool over that obtained with native serum but was 20% less effective in stimulating [35S]sulphate incorporation than the latter. Prior dialysis of serum had no effect on its modulatory action on either [35S]sulphate incorporation or on the size of UDP-sugar pools. 5. Insulin-like growth factor 1 (IGF-1), transforming growth factor beta-1 (TGF beta-1), platelet-derived growth factor (PDGF) (BB homodimer) and epidermal growth factor (EGF) all stimulated [35S]sulphate incorporation into glycosaminoglycans as expected. The UDP-glucuronate pool expanded by 1.5- and 2.0-fold in the presence of IGF-1 and TGF beta-1 respectively, and by about 1.8-fold in the presence of PDGF or EGF. None of the factors investigated, or combinations of IGF-1 and TGF beta-1 or IGF-1 and EGF, stimulated expansion of the UDP-glucuronate pool to the same extent as native serum.(ABSTRACT TRUNCATED AT 400 WORDS)
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Kotake, Toshihisa, Daisuke Yamaguchi, Hiroshi Ohzono, Sachiko Hojo, Satoshi Kaneko, Hide-ki Ishida, and Yoichi Tsumuraya. "UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts." Journal of Biological Chemistry 279, no. 44 (August 23, 2004): 45728–36. http://dx.doi.org/10.1074/jbc.m408716200.
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UDP-sugars, activated forms of monosaccharides, are synthesized throughde novoand salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativumL.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5–7.5, and at 45 °C in the presence of 2 mmMg2+. The apparentKmvalues for Glc 1-phosphate andl-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively.PsUSPcDNA was cloned by reverse transcriptase-PCR.PsUSPappears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues fromArabidopsisand rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed inEscherichia colicatalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.
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RANCOUR, David M., and Anant K. MENON. "Identification of endoplasmic reticulum proteins involved in glycan assembly: synthesis and characterization of P3-(4-azidoanilido)uridine 5′-triphosphate, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins." Biochemical Journal 333, no. 3 (August 1, 1998): 661–69. http://dx.doi.org/10.1042/bj3330661.
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Much of the enzymic machinery required for the assembly of cell surface carbohydrates is located in the endoplasmic reticulum (ER) of eukaryotic cells. Structural information on these proteins is limited and the identity of the active polypeptide(s) is generally unknown. This paper describes the synthesis and characteristics of a photoaffinity reagent that can be used to identify and analyse members of the ER glycan assembly apparatus, specifically those glycosyltransferases, nucleotide phosphatases and nucleotide–sugar transporters that recognize uridine nucleotides or UDP-sugars. The photoaffinity reagent, P3-(4-azidoanilido)uridine 5´-triphosphate (AAUTP), was synthesized easily from commercially available precursors. AAUTP inhibited the activity of ER glycosyltransferases that utilize UDP-GlcNAc and UDP-Glc, indicating that it is recognized by UDP-sugar-binding proteins. In preliminary tests AAUTP[α-32P] labelled bovine milk galactosyltransferase, a model UDP-sugar-utilizing enzyme, in a UV-light-dependent, competitive and saturable manner. When incubated with rat liver ER vesicles, AAUTP[α-32P] labelled a discrete subset of ER proteins; labelling was light-dependent and metal ion-specific. Photolabelling of intact ER vesicles with AAUTP[α-32P] caused selective incorporation of radioactivity into proteins with cytoplasmically disposed binding sites; UDP-Glc:glycoprotein glucosyltransferase, a lumenal protein, was labelled only when the vesicle membrane was disrupted. These data indicate that AAUTP is a membrane topological probe of catalytic sites in target proteins. Strategies for using AAUTP to identify and study novel ER proteins involved in glycan assembly are discussed.
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Zimmer, Brenna M., Joseph J. Barycki, and Melanie A. Simpson. "Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase." Journal of Histochemistry & Cytochemistry 69, no. 1 (August 4, 2020): 13–23. http://dx.doi.org/10.1369/0022155420947500.
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Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:
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Gu, Xiaogang, Sung G. Lee, and Maor Bar-Peled. "Biosynthesis of UDP-xylose and UDP-arabinose in Sinorhizobium meliloti 1021: first characterization of a bacterial UDP-xylose synthase, and UDP-xylose 4-epimerase." Microbiology 157, no. 1 (January 1, 2011): 260–69. http://dx.doi.org/10.1099/mic.0.040758-0.
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Sinorhizobium meliloti is a soil bacterium that fixes nitrogen after being established inside nodules that can form on the roots of several legumes, including Medicago truncatula. A mutation in an S. meliloti gene (lpsB) required for lipopolysaccharide synthesis has been reported to result in defective nodulation and an increase in the synthesis of a xylose-containing glycan. Glycans containing xylose as well as arabinose are also formed by other rhizobial species, but little is known about their structures and the biosynthetic pathways leading to their formation. To gain insight into the biosynthesis of these glycans and their biological roles, we report the identification of an operon in S. meliloti 1021 that contains two genes encoding activities not previously described in bacteria. One gene encodes a UDP-xylose synthase (Uxs) that converts UDP-glucuronic acid to UDP-xylose, and the second encodes a UDP-xylose 4-epimerase (Uxe) that interconverts UDP-xylose and UDP-arabinose. Similar genes were also identified in other rhizobial species, including Rhizobium leguminosarum, suggesting that they have important roles in the life cycle of this agronomically important class of bacteria. Functional studies established that recombinant SmUxs1 is likely to be active as a dimer and is inhibited by NADH and UDP-arabinose. SmUxe is inhibited by UDP-galactose, even though this nucleotide sugar is not a substrate for the 4-epimerase. Unambiguous evidence for the conversions of UDP-glucuronic acid to UDP-α-d-xylose and then to UDP-β-l-arabinose (UDP-arabinopyranose) was obtained using real-time 1H-NMR spectroscopy. Our results provide new information about the ability of rhizobia to form UDP-xylose and UDP-arabinose, which are then used for the synthesis of xylose- and arabinose-containing glycans.
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Guo, Yuxi, Junqiang Fang, Tiehai Li, Xu Li, Cheng Ma, Xuan Wang, Peng G. Wang, and Lei Li. "Comparing substrate specificity of two UDP-sugar pyrophosphorylases and efficient one-pot enzymatic synthesis of UDP-GlcA and UDP-GalA." Carbohydrate Research 411 (June 2015): 1–5. http://dx.doi.org/10.1016/j.carres.2015.04.001.
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Leroux, Mélanie, Julie Michaud, Eric Bayma, Sylvie Armand, Sophie Drouillard, and Bernard Priem. "Misincorporation of Galactose by Chondroitin Synthase of Escherichia coli K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide." Biomolecules 10, no. 12 (December 12, 2020): 1667. http://dx.doi.org/10.3390/biom10121667.
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Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated β3N-acetylgalactosamine (GalNAc)-β4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose β-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated β3-galactose (Gal)-β4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.
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Weigel, Paul H. "Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior." International Journal of Cell Biology 2015 (September 10, 2015): 1–15. http://dx.doi.org/10.1155/2015/367579.
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Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.
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Yang, Ting, and Maor Bar-Peled. "Identification of a novel UDP-sugar pyrophosphorylase with a broad substrate specificity in Trypanosoma cruzi." Biochemical Journal 429, no. 3 (July 14, 2010): 533–43. http://dx.doi.org/10.1042/bj20100238.
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The diverse types of glycoconjugates synthesized by trypanosomatid parasites are unique compared with the host cells. These glycans are required for the parasite survival, invasion or evasion of the host immune system. Synthesis of those glycoconjugates requires a constant supply of nucleotide-sugars (NDP-sugars), yet little is known about how these NDP-sugars are made and supplied. In the present paper, we report a functional gene from Trypanosoma cruzi that encodes a nucleotidyltransferase, which is capable of transforming different types of sugar 1-phosphates and NTP into NDP-sugars. In the forward reaction, the enzyme catalyses the formation of UDP-glucose, UDP-galactose, UDP-xylose and UDP-glucuronic acid, from their respective monosaccharide 1-phosphates in the presence of UTP. The enzyme could also convert glucose 1-phosphate and TTP into TDP-glucose, albeit at lower efficiency. The enzyme requires bivalent ions (Mg2+ or Mn2+) for its activity and is highly active between pH 6.5 and pH 8.0, and at 30–42 °C. The apparent Km values for the forward reaction were 177 μM (glucose 1-phosphate) and 28.4 μM (UTP) respectively. The identification of this unusual parasite enzyme with such broad substrate specificities suggests an alternative pathway that might play an essential role for nucleotide-sugar biosynthesis and for the regulation of the NDP-sugar pool in the parasite.
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Lee, Soon Goo, Eitan Salomon, Oliver Yu, and Joseph M. Jez. "Molecular basis for branched steviol glucoside biosynthesis." Proceedings of the National Academy of Sciences 116, no. 26 (June 10, 2019): 13131–36. http://dx.doi.org/10.1073/pnas.1902104116.
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Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 fromStevia rebaudianacatalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3′ position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.
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Vigetti, Davide, Sara Deleonibus, Eugenia Karousou, Manuela Viola, Giancarlo De Luca, and Alberto Passi. "Hyaluronan Produced by Smooth Muscle Cells Plays a Critical Role in Neointima Formation." Conference Papers in Science 2014 (May 12, 2014): 1–5. http://dx.doi.org/10.1155/2014/408427.
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Large body of evidence supports the idea that microenvironment plays a critical role in several pathologies including atherosclerosis and cancer. The amount of hyaluronan (HA) is involved in the microenvironment alterations and the concentration of this polymer reflects the progression of the diseases promoting neoangiogenesis, cell migration, and inflammation. The HA synthesis is regulated by several factors: UDP sugar precursors availability and the phosphorylation of synthetic enzyme HAS2 as well as specific drugs reducing the UDP precursors. The HAS2 phosphorylation is done by AMP kinase, a sensor of cell energy. When the cells have low energy, AMP kinase is activated and modifies covalently the regulatory enzymes, blocking all biosynthetic processes and activating the energy producing metabolism. It was recently reported that the hexosamine biosynthetic pathway (HBP) may increase the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. We demonstrated that the increase of HA synthesis depends on the HAS2 post translational modification O-GlcNAcylation, which increases HA secretion modifying a residue different from the phosphorylation site of AMP kinase. In this report we highlighted the critical aspects of the post translational HAS2 regulation and its influence on HA synthesis.
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Sosicka, Paulina, Bożena Bazan, Dorota Maszczak-Seneczko, Yauhen Shauchuk, Teresa Olczak, and Mariusz Olczak. "SLC35A5 Protein—A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity." International Journal of Molecular Sciences 20, no. 2 (January 11, 2019): 276. http://dx.doi.org/10.3390/ijms20020276.
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Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.
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Li, Z., L. Gao, Y. T. Wang, W. Zhu, J. L. Ye, and G. H. Li. "Carbohydrate Metabolism Changes in Prunus persica Gummosis Infected with Lasiodiplodia theobromae." Phytopathology® 104, no. 5 (May 2014): 445–52. http://dx.doi.org/10.1094/phyto-01-13-0025-r.
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Peach gummosis represents a significant global disease of stone fruit trees and a major disease in the south peach production area of the Yangtze River of China. In this study, the carbohydrate composition of peach shoots during infection by Lasiodiplodia theobromae was examined. The expression of genes related to metabolic enzymes was also investigated. Control wounded and noninoculated tissue, lesion tissue, and wounded and inoculated surrounding lesion tissue of peach shoots were analyzed. Soluble sugars, glucose, mannose, arabinose, and xylose significantly increased in inoculated tissues of peach shoots compared with control tissues at different times after inoculation. Accumulation of polysaccharides was also observed by section observation and periodic acid Schiff's reagent staining during infection. Analysis using quantitative reverse-transcription polymerase chain reaction revealed that the abundance of key transcripts on the synthesis pathway of uridine diphosphate (UDP)-D-glucuronate, UDP-D-galactose, and UDP-D-arabinose increased but the synthesis of L-galactose and guanosine diphosphate-L-galactose were inhibited. After inoculation, the transcript levels of sugar transport-related genes (namely, SUT, SOT, GMT, and UGT) was induced. These changes in sugar content and gene expression were directly associated with peach gum polysaccharide formation and may be responsible for the symptoms of peach gummosis.
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Zang, L. X., R. R. Du, H. C. Zang, F. S. Wang, and J. Z. Sheng. "Production of Arabidopsis thaliana UDP-Sugar Pyrophosphorylase by Pichia pastoris and Its Application in Efficient UDP-Glucose and UDP-Glucuronic Acid Synthesis." Applied Biochemistry and Microbiology 55, no. 6 (November 2019): 631–38. http://dx.doi.org/10.1134/s0003683819060152.
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Morii, Hiroyuki, Tadashi Eguchi, and Yosuke Koga. "In Vitro Biosynthesis of Ether-Type Glycolipids in the Methanoarchaeon Methanothermobacter thermautotrophicus." Journal of Bacteriology 189, no. 11 (April 6, 2007): 4053–61. http://dx.doi.org/10.1128/jb.01875-06.
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ABSTRACT The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the β-d-glucosyl-(1→6)-d-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose β-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose β-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose → MGA + UDP, and (ii) MGA + UDP-glucose → DGA + UDP. Both enzymes required the addition of K+ ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl2, in contrast to MGA synthase, which did not require Mg2+. It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg2+ requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg2+ requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg2+ increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.
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Kärkönen, Anna, Alain Murigneux, Jean-Pierre Martinant, Elodie Pepey, Christophe Tatout, Bernard J. Dudley, and Stephen C. Fry. "UDP-glucose dehydrogenases of maize: a role in cell wall pentose biosynthesis." Biochemical Journal 391, no. 2 (October 10, 2005): 409–15. http://dx.doi.org/10.1042/bj20050800.
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UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60–70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 μM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having Km values of approx. 380 and 950 μM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.
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Ramos, Ana, Ingeborg C. Boels, Willem M. de Vos, and Helena Santos. "Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis." Applied and Environmental Microbiology 67, no. 1 (January 1, 2001): 33–41. http://dx.doi.org/10.1128/aem.67.1.33-41.2001.
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ABSTRACT The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.
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Castro, Olga, Ling Yun Chen, Armando J. Parodi, and Claudia Abeijón. "Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum ofSaccharomyces cerevisiae:In Vivo and In Vitro Evidence." Molecular Biology of the Cell 10, no. 4 (April 1999): 1019–30. http://dx.doi.org/10.1091/mbc.10.4.1019.
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It has been proposed that synthesis of β-1,6-glucan, one ofSaccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparentKmof 46 μM and a Vmaxof 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) inS. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected inalg6 or alg5 mutant cells, which transfer Man9GlcNAc2to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.
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Muthana, Musleh M., Jingyao Qu, Yanhong Li, Lei Zhang, Hai Yu, Li Ding, Hamed Malekan, and Xi Chen. "Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)." Chemical Communications 48, no. 21 (2012): 2728. http://dx.doi.org/10.1039/c2cc17577k.
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Greig, Kylie T., Jennifer Antonchuk, Donald Metcalf, Phillip O. Morgan, Danielle L. Krebs, Jian-Guo Zhang, Douglas F. Hacking, et al. "Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development." Molecular and Cellular Biology 27, no. 16 (June 4, 2007): 5849–59. http://dx.doi.org/10.1128/mcb.00802-07.
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ABSTRACT Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity.
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Choi, Yong Kee, Yoon Kyung Hwang, Yong Han Kang, and Young Shik Park. "Chemical structure of 1-O-(L-erythro-biopterin-2'-yl)-a-glucose isolated from a cyanobacterium Synechococcus sp. PCC 7942." Pteridines 12, no. 3 (August 2001): 121–25. http://dx.doi.org/10.1515/pteridines.2001.12.3.121.
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Abstract A pteridine glycoside in Synechococcus sp. PCC 7942, the structure of which had been tentatively identified as biopterin-glucoside, was isolated and characterized for its exact chemical structure by 2D-NMR spectroscopy. The determined structure is 1-0-(L-erythro-biopterin-2'-yl)-α-glucose. It is the first report on the occurrence of a biopterin-glucoside having a α-configured sugar directly attached at the pteridine ring. This result also supports that the previously purified UDP-glucose: BH4 glucosyltransferase from Synechococcus sp. PCC 7942, which catalyzes the synthesis of BH4-glucoside from UDP-glucose and BH4, is a α-glucosyltransferase.
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Sharma, Monica, Swati Sharma, Pallab Ray, and Anuradha Chakraborti. "Targeting Streptococcus pneumoniae UDP-glucose pyrophosphorylase (UGPase): in vitro validation of a putative inhibitor." Drug Target Insights 14, no. 1 (October 7, 2020): 26–33. http://dx.doi.org/10.33393/dti.2020.2103.
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Background: Genome plasticity of Streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide based vaccines. Therefore targets independent of capsular types are sought to control the pneumococcal pathogenicity. UcrDP-glucose pyrophosphorylase (UGPase) is one such desired candidate being responsible for the synthesis of UDP-glucose, a sugar-precursor in capsular biosynthesis and metabolic Leloir pathway. Being crucial to pneumococcal pathobiology, the effect of UGPase inhibition on virulence was evaluated in vitro.
Methods: A putative inhibitor (UDP) was evaluated for effective inhibitory concentration in S. pneumoniae and A549 cells, its efficacy and toxicity. Effect of UDP on adherence and phagocytosis was measured in human respiratory epithelial (A549 and HEp-2) and macrophage (THP1 and J774.A.1) cell lines respectively.
Results: A differential effective inhibitory concentration of UDP for UGPase inhibition was observed in S. pneumoniae and A549 cells i.e. 5 µM and 100 µM respectively. UDP treatments lowered percent cytotoxicity in pneumococcal infected monolayers and didn't exert adverse effects on viabilities. S. pneumoniae adherence to host cells was decreased significantly with UDP treatments. UDP induced the secretion of IL-1β, TNF-α, IL-6, and IL-8 and increased pneumococcal phagocytosis.
Conclusion: Our study shows UDP mediated decrease in the virulence of S. pneumoniae and demonstrates UDP as an effective inhibitor of pneumococcal UGPase.
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Szulc, Bozena, Paulina Sosicka, Dorota Maszczak-Seneczko, Edyta Skurska, Auhen Shauchuk, Teresa Olczak, Hudson H. Freeze, and Mariusz Olczak. "Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3." Journal of Biological Chemistry 295, no. 48 (September 16, 2020): 16445–63. http://dx.doi.org/10.1074/jbc.ra119.012362.
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Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.
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Silva, Elisabete, Ana Rita Marques, Arsénio Mendes Fialho, Ana Teresa Granja, and Isabel Sá-Correia. "Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities." Applied and Environmental Microbiology 71, no. 8 (August 2005): 4703–12. http://dx.doi.org/10.1128/aem.71.8.4703-4712.2005.
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ABSTRACT The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).
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Cartee, Robert T., W. Thomas Forsee, and Janet Yother. "Initiation and Synthesis of the Streptococcus pneumoniae Type 3 Capsule on a Phosphatidylglycerol Membrane Anchor." Journal of Bacteriology 187, no. 13 (July 1, 2005): 4470–79. http://dx.doi.org/10.1128/jb.187.13.4470-4479.2005.
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ABSTRACT The type 3 synthase from Streptococcus pneumoniae is a processive β-glycosyltransferase that assembles the type 3 polysaccharide [3)-β-d-GlcUA-(1→4)-β-d-Glc-(1→] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[14C]Glc or UDP-[14C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.
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Westman, Erin L., David J. Mcnally, Martin Rejzek, Wayne L. Miller, Vellupillai Sri Kannathasan, Andrew Preston, Duncan J. Maskell, Robert A. Field, Jean-Robert Brisson, and Joseph S. Lam. "Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases from respiratory pathogens." Biochemical Journal 405, no. 1 (June 13, 2007): 123–30. http://dx.doi.org/10.1042/bj20070017.
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The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6–WbpI and His6–WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6–WbpI or His6–WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme–substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6–WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases.
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Muthana, Musleh M., Jingyao Qu, Yanhong Li, Lei Zhang, Hai Yu, Li Ding, Hamed Malekan, and Xi Chen. "ChemInform Abstract: Efficient One-Pot Multienzyme Synthesis of UDP-Sugars Using a Promiscuous UDP-Sugar Pyrophosphorylase from Bifidobacterium longum (BLUSP)." ChemInform 43, no. 26 (May 31, 2012): no. http://dx.doi.org/10.1002/chin.201226210.
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Paczkowski, C., M. Kalinowska, and Z. A. Wojciechowski. "UDP-glucose:solasodine glucosyltransferase from eggplant (Solanum melongena L.) leaves: partial purification and characterization." Acta Biochimica Polonica 44, no. 1 (March 31, 1997): 43–53. http://dx.doi.org/10.18388/abp.1997_4438.
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Uridine 5'-diphosphoglucose-dependent glucosyltransferase which catalyzes the glycosylation of solasodine i.e. UDP-glucose:solasodine glucosyltransferase, is present in leaves, roots, unripe fruits and unripe seeds of eggplant (Solanum melongena L.). The glucosylation product is chromatographically identical with authentic solasodine 3 beta-D-monoglucoside, a putative intermediate in the biosynthesis of solasodine-based glycoalkaloids characteristic of the eggplant. The enzyme was purified about 50-fold from crude cytosol fraction of eggplant leaves by ammonium sulphate precipitation and column chromatography on Q-Sepharose and Sephadex G-100. The native enzyme has a molecular mass of approx. 55 kDa and pH optimum of 8.5. Divalent metal ions are not required for its activity but the presence of free-SH groups is essential. Besides solasodine (Km = 0.04 microM), the enzyme effectively glucosylates tomatidine, another steroidal alkaloid of the spirosolane type, but it is virtually inactive towards the solanidane-type steroidal alkaloids such as solanidine or demissidine. The enzyme is specific for UDP-glucose (Km = 2.1 microM) since unlabelled ADP-, GDP-, CDP- or TDP-glucose could not effectively compete with UDP-[14C]glucose used as the sugar donor for solasodine glucosylation. Moreover, no synthesis of labelled solasodine galactoside was observed when UDP-[14C]glucose was replaced with UDP-[14C]galactose.
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Beverley, Stephen M., Katherine L. Owens, Melissa Showalter, Cara L. Griffith, Tamara L. Doering, Victoria C. Jones, and Michael R. McNeil. "Eukaryotic UDP-Galactopyranose Mutase (GLF Gene) in Microbial and Metazoal Pathogens." Eukaryotic Cell 4, no. 6 (June 2005): 1147–54. http://dx.doi.org/10.1128/ec.4.6.1147-1154.2005.
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ABSTRACT Galactofuranose (Gal f ) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Gal f is synthesized as the nucleotide sugar UDP-Gal f by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Gal f synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Gal f synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Gal f synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Gal f plays critical roles in cellular viability and virulence.
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Xu, De-Qi, John Thompson, and John O. Cisar. "Genetic Loci for Coaggregation Receptor Polysaccharide Biosynthesis in Streptococcus gordonii 38." Journal of Bacteriology 185, no. 18 (September 15, 2003): 5419–30. http://dx.doi.org/10.1128/jb.185.18.5419-5430.2003.
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ABSTRACT The cell wall polysaccharide of Streptococcus gordonii 38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAcβ1→3Gal-containing recognition motif. The same RPS has now been identified from S. gordonii AT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize the S. gordonii 38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designated galE1, was identified as a pseudogene in the galactose operon, and the second, designated galE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e., rmlA, rmlC, and rmlB). Insertional inactivation of galE2 abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of the galE1 pseudogene in this galE2 mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production by S. gordonii 38 depends on the dual specificity of the epimerase encoded by galE2.
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Wong, Chi Huey, Ruo Wang, and Yoshitaka Ichikawa. "Regeneration of sugar nucleotide for enzymic oligosaccharide synthesis: use of Gal-1-phosphate uridyltransferase in the regeneration of UDP-galactose, UDP-2-deoxygalactose, and UDP-galactosamine." Journal of Organic Chemistry 57, no. 16 (July 1992): 4343–44. http://dx.doi.org/10.1021/jo00042a008.
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Capul, Althea A., Tamara Barron, Deborah E. Dobson, Salvatore J. Turco, and Stephen M. Beverley. "Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis inLeishmania major." Journal of Biological Chemistry 282, no. 19 (March 8, 2007): 14006–17. http://dx.doi.org/10.1074/jbc.m610869200.
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Mikušová, Katarína, Martina Beláňová, Jana Korduláková, Kristine Honda, Michael R. McNeil, Sebabrata Mahapatra, Dean C. Crick, and Patrick J. Brennan. "Identification of a Novel Galactosyl Transferase Involved in Biosynthesis of the Mycobacterial Cell Wall." Journal of Bacteriology 188, no. 18 (September 15, 2006): 6592–98. http://dx.doi.org/10.1128/jb.00489-06.
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ABSTRACT The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the “possible arabinogalactan biosynthetic gene cluster” of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C50-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[14C]galactopyranose as the immediate precursor of UDP-[14C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C50-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C50-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C50-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.
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Bengoechea, José Antonio, Elise Pinta, Tiina Salminen, Clemens Oertelt, Otto Holst, Joanna Radziejewska-Lebrecht, Zofia Piotrowska-Seget, Reija Venho, and Mikael Skurnik. "Functional Characterization of Gne (UDP-N-Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of Yersinia enterocolitica Serotype O:8." Journal of Bacteriology 184, no. 15 (August 1, 2002): 4277–87. http://dx.doi.org/10.1128/jb.184.15.4277-4287.2002.
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ABSTRACT The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), l-fucose (Fuc), d-galactose (Gal), d-mannose (Man), and 6-deoxy-d-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.
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KOSTOVA, Zlatka, David M. RANCOUR, Anant K. MENON, and Peter ORLEAN. "Photoaffinity labelling with P3-(4-azidoanilido)uridine 5′-triphosphate identifies Gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis." Biochemical Journal 350, no. 3 (September 8, 2000): 815–22. http://dx.doi.org/10.1042/bj3500815.
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Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes. The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI). Four proteins in mammals and at least three in yeast make up a complex that carries out this reaction. Three of the proteins are highly conserved between yeast and mammals: the Gpi1 protein, the Pig-C/Gpi2 protein and the Pig-A/Gpi3 protein. The function of the individual subunits is not known, but of the three, the Pig-A/Gpi3 proteins resemble members of a large family of nucleotide-sugar-utilizing glycosyltransferases. To establish whether Gpi3p is the UDP-GlcNAc-binding subunit of the yeast GlcNAc-PI synthetic complex, we tested its ability to become cross-linked to the photoactivatable substrate analogue P3-(4-azidoanilido)-uridine 5´-triphosphate (AAUTP). We report that Gpi3p bearing the FLAG epitope at its C-terminus becomes cross-linked to AAUTP[α-32P], but that Gpi2p-FLAG does not. Furthermore, Gpi3p-FLAG expressed in Escherichiacoli is also cross-linked. These results indicate that Gpi3p is the UDP-GlcNAc-binding and probable catalytic subunit of the GlcNAc-PI synthetic complex.
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Chapeau, Marie-Christine, and Perry A. Frey. "Synthesis of UDP-4-deoxy-4-fluoroglucose and UDP-4-deoxy-4-fluorogalactose and their Interactions with Enzymes of Nucleotide Sugar Metabolism." Journal of Organic Chemistry 59, no. 23 (November 1994): 6994–98. http://dx.doi.org/10.1021/jo00102a024.
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DeVito, Stefanie Renee, Emilio Ortiz-Riaño, Luis Martínez-Sobrido, and Joshua Munger. "Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP–sugar synthesis to support viral protein glycosylation." Proceedings of the National Academy of Sciences 111, no. 50 (December 3, 2014): 18019–24. http://dx.doi.org/10.1073/pnas.1415864111.
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Mian, N. "Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex." Biochemical Journal 237, no. 2 (July 15, 1986): 343–57. http://dx.doi.org/10.1042/bj2370343.
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A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.
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SUGUMARAN, Geetha, Maya KATSMAN, and E. Jeremiah SILBERT. "Subcellular co-localization and potential interaction of glucuronosyltransferases with nascent proteochondroitin sulphate at Golgi sites of chondroitin synthesis." Biochemical Journal 329, no. 1 (January 1, 1998): 203–8. http://dx.doi.org/10.1042/bj3290203.
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Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(β1-3)Galβ1-methyl (disaccharide) and GalNAc(β1-4)GlcA(β1-3)GalNAc(β1-4)GlcA(β1-3)GalNAc (pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.
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Ravishankar, Sudha, Vidya Prasanna Kumar, B. Chandrakala, Ramesh K. Jha, Suresh M. Solapure, and Sunita M. de Sousa. "Scintillation Proximity Assay for Inhibitors of Escherichia coli MurG and, Optionally, MraY." Antimicrobial Agents and Chemotherapy 49, no. 4 (April 2005): 1410–18. http://dx.doi.org/10.1128/aac.49.4.1410-1418.2005.
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ABSTRACT MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis—penicillin G, moenomycin, and bacitracin—had no effect. Surprisingly, however, the β-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.
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Liu, Song Yu, and John P. N. Rosazza. "Enzymatic Conversion of Glucose to UDP-4-Keto-6-Deoxyglucose in Streptomyces spp." Applied and Environmental Microbiology 64, no. 10 (October 1, 1998): 3972–76. http://dx.doi.org/10.1128/aem.64.10.3972-3976.1998.
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ABSTRACT All of the 2,6-dideoxy sugars contained within the structure of chromomycin A3 are derived from d-glucose. Enzyme assays were used to confirm the presence of hexokinase, phosphoglucomutase, UDPG pyrophosphorylase (UDPGP), and UDPG oxidoreductase (UDPGO), all of which are involved in the pathway of glucose activation and conversion into 2,6-dideoxyhexoses during chromomycin biosynthesis. Levels of the four enzymes inStreptomyces spp. cell extracts were correlated with the production of chromomycins. The pathway of sugar activation inStreptomyces spp. involves glucose 6-phosphorylation by hexokinase, isomerization to G-1-P catalyzed by phosphoglucomutase, synthesis of UDPG catalyzed by UDPGP, and formation of UDP-4-keto-6-deoxyglucose by UDPGO.
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Yang, Ting, Merritt Echols, Andy Martin, and Maor Bar-Peled. "Identification and characterization of a strict and a promiscuous N-acetylglucosamine-1-P uridylyltransferase in Arabidopsis." Biochemical Journal 430, no. 2 (August 13, 2010): 275–84. http://dx.doi.org/10.1042/bj20100315.
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UDP-GlcNAc is an essential precursor for glycoprotein and glycolipid synthesis. In the present study, a functional nucleotidyltransferase gene from Arabidopsis encoding a 58.3 kDa GlcNAc1pUT-1 (N-acetylglucosamine-1-phosphate uridylyltransferase) was identified. In the forward reaction the enzyme catalyses the formation of UDP-N-acetylglucosamine and PPi from the respective monosaccharide 1-phosphate and UTP. The enzyme can utilize the 4-epimer UDP-GalNAc as a substrate as well. The enzyme requires divalent ions (Mg2+ or Mn2+) for activity and is highly active between pH 6.5 and 8.0, and at 30–37 °C. The apparent Km values for the forward reaction were 337 μM (GlcNAc-1-P) and 295 μM (UTP) respectively. Another GlcNAc1pUT-2, which shares 86% amino acid sequence identity with GlcNAc1pUT-1, was found to convert, in addition to GlcNAc-1-P and GalNAc-1-P, Glc-1-P into corresponding UDP-sugars, suggesting that subtle changes in the UT family cause different substrate specificities. A three-dimensional protein structure model using the human AGX1 as template showed a conserved catalytic fold and helped identify key conserved motifs, despite the high sequence divergence. The identification of these strict and promiscuous gene products open a window to indentify new roles of amino sugar metabolism in plants and specifically their role as signalling molecules. The ability of GlcNAc1pUT-2 to utilize three different substrates may provide further understanding as to why biological systems have plasticity.