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1
Li, Zijie, Yahui Gao, Hideki Nakanishi, Xiaodong Gao, and Li Cai. "Biosynthesis of rare hexoses using microorganisms and related enzymes." Beilstein Journal of Organic Chemistry 9 (November 12, 2013): 2434–45. http://dx.doi.org/10.3762/bjoc.9.281.
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Rare sugars, referred to as monosaccharides and their derivatives that rarely exist in nature, can be applied in many areas ranging from foodstuffs to pharmaceutical and nutrition industry, or as starting materials for various natural products and drug candidates. Unfortunately, an important factor restricting the utilization of rare sugars is their limited availability, resulting from limited synthetic methods. Nowadays, microbial and enzymatic transformations have become a very powerful tool in this field. This article reviews the biosynthesis and enzymatic production of rare ketohexoses, aldohexoses and sugar alcohols (hexitols), including D-tagatose, D-psicose, D-sorbose, L-tagatose, L-fructose, 1-deoxy-L-fructose, D-allose, L-glucose, L-talose, D-gulose, L-galactose, L-fucose, allitol, D-talitol, and L-sorbitol. New systems and robust catalysts resulting from advancements in genomics and bioengineering are also discussed.
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Begander, Benjamin, Anna Huber, Josef Sperl, and Volker Sieber. "Development of a Cofactor Balanced, Multi Enzymatic Cascade Reaction for the Simultaneous Production of L-Alanine and L-Serine from 2-Keto-3-deoxy-gluconate." Catalysts 11, no. 1 (December 30, 2020): 31. http://dx.doi.org/10.3390/catal11010031.
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Enzymatic reaction cascades represent a powerful tool to convert biogenic resources into valuable chemicals for fuel and commodity markets. Sugars and their breakdown products constitute a significant group of possible substrates for such biocatalytic conversion strategies to value-added products. However, one major drawback of sugar cascades is the need for cofactor recycling without using additional enzymes and/or creating unwanted by-products. Here, we describe a novel, multi-enzymatic reaction cascade for the one-pot simultaneous synthesis of L-alanine and L-serine, using the sugar degradation product 2-keto-3-deoxygluconate and ammonium as precursors. To pursue this aim, we used four different, thermostable enzymes, while the necessary cofactor NADH is recycled entirely self-sufficiently. Buffer and pH optimisation in combination with an enzyme titration study yielded an optimised production of 21.3 +/− 1.0 mM L-alanine and 8.9 +/− 0.4 mM L-serine in one pot after 21 h.
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Sutiono, Samuel, Bettina Siebers, and Volker Sieber. "Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals." Applied Microbiology and Biotechnology 104, no. 16 (June 21, 2020): 7023–35. http://dx.doi.org/10.1007/s00253-020-10742-5.
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Abstract2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points• L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively.• Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs.• CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield.• Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.
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Kirby, James, Minobu Nishimoto, Ruthie W. N. Chow, Edward E. K. Baidoo, George Wang, Joel Martin, Wendy Schackwitz, Rossana Chan, Jeffrey L. Fortman, and Jay D. Keasling. "Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate." Applied and Environmental Microbiology 81, no. 1 (October 17, 2014): 130–38. http://dx.doi.org/10.1128/aem.02920-14.
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ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.
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Shibaev, V. N., and L. L. Danilov. "New developments in the synthesis of phosphopolyprenols and their glycosyl esters." Biochemistry and Cell Biology 70, no. 6 (June 1, 1992): 429–37. http://dx.doi.org/10.1139/o92-066.
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Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of α- and β-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.Key words: polyprenyl phosphates, polyprenyl phosphosugars, chemical phosphorylation, specificity of enzymes.
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Zelent, Bogumil, Stella Odili, Carol Buettger, Chiyo Shiota, Joseph Grimsby, Rebecca Taub, Mark A. Magnuson, Jane M. Vanderkooi, and Franz M. Matschinsky. "Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence." Biochemical Journal 413, no. 2 (June 26, 2008): 269–80. http://dx.doi.org/10.1042/bj20071718.
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Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST–GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (ϕ) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST–GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose≫L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp99 and Trp167 were large compared with that from Trp257 and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.
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Voitsekhovskaia, Irina, Constanze Paulus, Charlotte Dahlem, Yuriy Rebets, Suvd Nadmid, Josef Zapp, Denis Axenov-Gribanov, et al. "Baikalomycins A-C, New Aquayamycin-Type Angucyclines Isolated from Lake Baikal Derived Streptomyces sp. IB201691-2A." Microorganisms 8, no. 5 (May 7, 2020): 680. http://dx.doi.org/10.3390/microorganisms8050680.
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Natural products produced by bacteria found in unusual and poorly studied ecosystems, such as Lake Baikal, represent a promising source of new valuable drug leads. Here we report the isolation of a new Streptomyces sp. strain IB201691-2A from the Lake Baikal endemic mollusk Benedictia baicalensis. In the course of an activity guided screening three new angucyclines, named baikalomycins A–C, were isolated and characterized, highlighting the potential of poorly investigated ecological niches. Besides that, the strain was found to accumulate large quantities of rabelomycin and 5-hydroxy-rabelomycin, known shunt products in angucyclines biosynthesis. Baikalomycins A–C demonstrated varying degrees of anticancer activity. Rabelomycin and 5-hydroxy-rabelomycin further demonstrated antiproliferative activities. The structure elucidation showed that baikalomycin A is a modified aquayamycin with β-d-amicetose and two additional hydroxyl groups at unusual positions (6a and 12a) of aglycone. Baikalomycins B and C have alternating second sugars attached, α-l-amicetose and α-l-aculose, respectively. The gene cluster for baikalomycins biosynthesis was identified by genome mining, cloned using a transformation-associated recombination technique and successfully expressed in S. albus J1074. It contains a typical set of genes responsible for an angucycline core assembly, all necessary genes for the deoxy sugars biosynthesis, and three genes coding for the glycosyltransferase enzymes. Heterologous expression and deletion experiments allowed to assign the function of glycosyltransferases involved in the decoration of baikalomycins aglycone.
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Zaret, K. S., and K. A. Stevens. "Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line." Molecular and Cellular Biology 10, no. 9 (September 1990): 4582–89. http://dx.doi.org/10.1128/mcb.10.9.4582.
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To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.
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Zaret, K. S., and K. A. Stevens. "Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line." Molecular and Cellular Biology 10, no. 9 (September 1990): 4582–89. http://dx.doi.org/10.1128/mcb.10.9.4582-4589.1990.
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To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.
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Rahman, Mohammad Mubinur, Martina Andberg, Anu Koivula, Juha Rouvinen, and Nina Hakulinen. "Crystallization and X-ray diffraction analysis of anL-arabinonate dehydratase fromRhizobium leguminosarumbv.trifoliiand aD-xylonate dehydratase fromCaulobacter crescentus." Acta Crystallographica Section F Structural Biology Communications 72, no. 8 (July 13, 2016): 604–8. http://dx.doi.org/10.1107/s2053230x16010311.
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L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase fromRhizobium leguminosarumbv.trifolii(RlArDHT) and D-xylonate dehydratase fromCaulobacter crescentus(CcXyDHT) were heterologously expressed inEscherichia coliand purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals ofRlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space groupP21, with unit-cell parametersa = 106.07,b= 208.61,c= 147.09 Å, β = 90.43°. EightRlArDHT molecules (two tetramers) in the asymmetric unit give aVMvalue of 3.2 Å3 Da−1and a solvent content of 62%. Crystals ofCcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space groupC2, with unit-cell parametersa= 270.42,b= 236.13,c = 65.17 Å, β = 97.38°. FourCcXyDHT molecules (a tetramer) in the asymmetric unit give aVMvalue of 4.0 Å3 Da−1and a solvent content of 69%.
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Shamsi Kazem Abadi, Saeideh, Matthew C. Deen, Jacqueline N. Watson, Fahimeh S. Shidmoossavee, and Andrew J. Bennet. "Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase." Glycobiology 30, no. 5 (December 4, 2019): 325–33. http://dx.doi.org/10.1093/glycob/cwz099.
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Abstract N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M−1 s−1; the best variant displayed an efficiency of >106 M−1 s−1 at its optimal pH.
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Varghese, Elizabeth, Samson Mathews Samuel, Alena Líšková, Marek Samec, Peter Kubatka, and Dietrich Büsselberg. "Targeting Glucose Metabolism to Overcome Resistance to Anticancer Chemotherapy in Breast Cancer." Cancers 12, no. 8 (August 12, 2020): 2252. http://dx.doi.org/10.3390/cancers12082252.
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Breast cancer (BC) is the most prevalent cancer in women. BC is heterogeneous, with distinct phenotypical and morphological characteristics. These are based on their gene expression profiles, which divide BC into different subtypes, among which the triple-negative breast cancer (TNBC) subtype is the most aggressive one. The growing interest in tumor metabolism emphasizes the role of altered glucose metabolism in driving cancer progression, response to cancer treatment, and its distinct role in therapy resistance. Alterations in glucose metabolism are characterized by increased uptake of glucose, hyperactivated glycolysis, decreased oxidative phosphorylation (OXPHOS) component, and the accumulation of lactate. These deviations are attributed to the upregulation of key glycolytic enzymes and transporters of the glucose metabolic pathway. Key glycolytic enzymes such as hexokinase, lactate dehydrogenase, and enolase are upregulated, thereby conferring resistance towards drugs such as cisplatin, paclitaxel, tamoxifen, and doxorubicin. Besides, drug efflux and detoxification are two energy-dependent mechanisms contributing to resistance. The emergence of resistance to chemotherapy can occur at an early or later stage of the treatment, thus limiting the success and outcome of the therapy. Therefore, understanding the aberrant glucose metabolism in tumors and its link in conferring therapy resistance is essential. Using combinatory treatment with metabolic inhibitors, for example, 2-deoxy-D-glucose (2-DG) and metformin, showed promising results in countering therapy resistance. Newer drug designs such as drugs conjugated to sugars or peptides that utilize the enhanced expression of tumor cell glucose transporters offer selective and efficient drug delivery to cancer cells with less toxicity to healthy cells. Last but not least, naturally occurring compounds of plants defined as phytochemicals manifest a promising approach for the eradication of cancer cells via suppression of essential enzymes or other compartments associated with glycolysis. Their benefits for human health open new opportunities in therapeutic intervention, either alone or in combination with chemotherapeutic drugs. Importantly, phytochemicals as efficacious instruments of anticancer therapy can suppress events leading to chemoresistance of cancer cells. Here, we review the current knowledge of altered glucose metabolism in contributing to resistance to classical anticancer drugs in BC treatment and various ways to target the aberrant metabolism that will serve as a promising strategy for chemosensitizing tumors and overcoming resistance in BC.
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Zou, Lu, Ruixiang Blake Zheng, and Todd L. Lowary. "Studies on the substrate specificity of a GDP-mannose pyrophosphorylase from Salmonella enterica." Beilstein Journal of Organic Chemistry 8 (August 1, 2012): 1219–26. http://dx.doi.org/10.3762/bjoc.8.136.
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A series of methoxy and deoxy derivatives of mannopyranose-1-phosphate (Manp-1P) were chemically synthesized, and their ability to be converted into the corresponding guanosine diphosphate mannopyranose (GDP-Manp) analogues by a pyrophosphorylase (GDP-ManPP) from Salmonella enterica was studied. Evaluation of methoxy analogues demonstrated that GDP-ManPP is intolerant of bulky substituents at the C-2, C-3, and C-4 positions, in turn suggesting that these positions are buried inside the enzyme active site. Additionally, both the 6-methoxy and 6-deoxy Manp-1P derivatives are good or moderate substrates for GDP-ManPP, thus indicating that the C-6 hydroxy group of the Manp-1P substrate is not required for binding to the enzyme. When taken into consideration with other previously published work, it appears that this enzyme has potential utility for the chemoenzymatic synthesis of GDP-Manp analogues, which are useful probes for studying enzymes that employ this sugar nucleotide as a substrate.
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Percival, M. David, and Stephen G. Withers. "Applications of enzymes in the synthesis and hydrolytic study of 2-deoxy-α-D-glucopyranosyl phosphate." Canadian Journal of Chemistry 66, no. 8 (August 1, 1988): 1970–72. http://dx.doi.org/10.1139/v88-317.
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The labile sugar phosphate 2-deoxy-α-D-glucopyranosyl phosphate has been synthesized enzymically in a two-step process from 2-deoxy-D-glucose-6-phosphate via an intermediate uridine 5′-diphospho-2-deoxy-D-glucose. Rate constants for acid-catalysed (1 M HClO4) hydrolysis at several temperatures were determined by using an enzymic assay to measure remaining substrate. The values obtained were consistent with that anticipated on the basis of known hydrolysis rates for alkyl- and aryl-2-deoxy-D-glucopyranosides.
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Shishmarev, Dmitry, Lucas Quiquempoix, Clément Q. Fontenelle, Bruno Linclau, and Philip W. Kuchel. "Anomerisation of Fluorinated Sugars by Mutarotase Studied Using 19F NMR Two-Dimensional Exchange Spectroscopy." Australian Journal of Chemistry 73, no. 3 (2020): 117. http://dx.doi.org/10.1071/ch19562.
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Five 19F-substituted glucose analogues were used to probe the activity and mechanism of the enzyme mutarotase by using magnetisation-exchange NMR spectroscopy. The sugars (2-fluoro-2-deoxy-d-glucose, FDG2; 3-fluoro-3-deoxy-d-glucose, FDG3; 4-fluoro-4-deoxy-d-glucose, FDG4; 2,3-difluoro-2,3-dideoxy-d-glucose, FDG23; and 2,2,3,3-tetrafluoro-2,3-dideoxy-d-glucose (2,3-dideoxy-2,2,3,3-tetrafluoro-d-erythro-hexopyranose), FDG2233) showed separate 19F NMR spectroscopic resonances from their respective α- and β-anomers, thus allowing two-dimensional exchange spectroscopy measurements of the anomeric interconversion at equilibrium, on the time scale of a few seconds. Mutarotase catalysed the rapid exchange between the anomers of FDG4, but not the other four sugars. This finding, combined with previous work identifying the mechanism of the anomerisation by mutarotase, suggests that the rotation around the C1–C2 bond of the pyranose ring is the rate-limiting reaction step. In addition to d-glucose itself, it was shown that all other fluorinated sugars inhibited the FDG4 anomerisation, with the tetrafluorinated FDG2233 being the most potent inhibitor. Inhibition of mutarotase by F-sugars paves the way for the development of novel fluorinated compounds that are able to affect the activity of this enzyme invitro and invivo.
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Li, Zi, Thiya Mukherjee, Kyle Bowler, Sholeh Namdari, Zachary Snow, Sarah Prestridge, Alexandra Carlton, and Maor Bar-Peled. "A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars." Journal of Biological Chemistry 292, no. 18 (March 15, 2017): 7636–50. http://dx.doi.org/10.1074/jbc.m117.777417.
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Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus.
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Boer, S. H. De, J. J. Bradshaw-Rouse, L. Sequeira, and M. E. McNaughton. "Sugar composition and serological specificity of Erwinia carotovora lipopolysaccharides." Canadian Journal of Microbiology 31, no. 7 (July 1, 1985): 583–86. http://dx.doi.org/10.1139/m85-110.
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Sugar composition and serological specificity of the lipopolysaccharides (LPS) purified from 16 Erwinia carotovora strains in six different serogroups were determined. All the LPS preparations contained 2-keto-3-deoxyoctonoic acid, glucosamine, heptose, and glucose, and most contained galactose. Either rhamnose or fucose was present in LPS from 12 of the strains, and the presence or absence of these deoxy sugars was consistent for all strains within a serogroup. LPS from two strains contained mannose. One unidentified sugar was present in all LPS preparations, but six other unidentified sugars varied in different LPS preparations. All LPS preparations reacted in an enzyme-linked, immunosorbent assay (ELISA) with antisera produced against whole cells of a type strain in the same serogroup as the strain from which the LPS was extracted. Several cross-reactions among strains that previously were observed in immunodiffusion tests with whole-cell preparations were also observed in ELISA with purified LPS. Some of the LPS preparations also reacted in immunodiffusion with a precipitin line of identity with whole-cell preparations. The results indicate that E. carotovora serogroups probably are based on the LPS 0-antigen.
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Rempel, Brian P., and Stephen G. Withers. "Non-Stick Sugars: Synthesis of Difluorosugar Fluorides as Potential Glycosidase Inactivators." Australian Journal of Chemistry 62, no. 6 (2009): 590. http://dx.doi.org/10.1071/ch09223.
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Four new difluorosugar fluorides, 2-deoxy-2,5-difluoro-α-l-idopyranosyl fluoride, 1,5-difluoro-d-glucopyranosyl fluoride, 1,5-difluoro-l-idopyranosyl fluoride, and 2-deoxy-1,2-difluoro-d-glucopyranosyl fluoride, were synthesized from known precursors by a radical bromination/fluoride displacement sequence, followed by deprotection. The compounds were tested as time-dependent inactivators of the β-glucosidase from Agrobacterium sp. (Abg, EC 3.2.1.21) and, while they were shown to bind to the enzyme active site as reversible competitive inhibitors, the only time-dependent inactivation observed was traced to the presence of an extremely small amount (<0.1%) of a highly reactive contaminating impurity.
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Stead, Christopher, An Tran, Donald Ferguson, Sara McGrath, Robert Cotter, and Stephen Trent. "A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide." Journal of Bacteriology 187, no. 10 (May 15, 2005): 3374–83. http://dx.doi.org/10.1128/jb.187.10.3374-3383.2005.
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ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.
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Daniellou, Richard, Hongyan Zheng, and David RJ Palmer. "Kinetics of the reaction catalyzed by inositol dehydrogenase from Bacillus subtilis and inhibition by fluorinated substrate analogs." Canadian Journal of Chemistry 84, no. 4 (April 1, 2006): 522–27. http://dx.doi.org/10.1139/v06-033.
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Inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis catalyzes the oxidation of myo-inositol to scyllo-inosose by transfer of the equatorial hydride of the substrate to NAD+. This is a key enzyme in the metabolism of myo-inositol, a primary carbon source for soil bacteria. In light of our recent discovery that the enzyme has a broad substrate spectrum while maintaining high stereoselectivity, we seek a more thorough understanding of the enzyme and its active site. We have examined the kinetics of the recombinant enzyme, and synthesized fluorinated substrate analogues as competitive inhibitors. We have evaluated all rate constants in the ordered, sequential Bi Bi mechanism. No steady-state kinetic isotope effect is observed using myo-[2-2H]-inositol, indicating that the chemical step of the reaction is not rate-limiting. We have synthesized the substrate analogs 2-deoxy-2-fluoro-myo-inositol, its equatorial analog 1-deoxy-1-fluoro-scyllo-inositol, the gem-difluorinated analog 1-deoxy-1,1-difluoro-scyllo-inositol, and the sugar analog α-D-glucosyl fluoride. Of these, 1-deoxy-1-fluoro-scyllo-inositol showed no inhibition, while all others tested had Ki values comparable to the Km values of the analogous substrates myo-inositol and α-D-glucose.Key words: inositol dehydrogenase, enzyme mechanism, kinetics, competitive inhibitor, substrate analogue.
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Wojciechowski, Marek, Sławomir Milewski, Jan Mazerski, and Edward Borowski. "Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design." Acta Biochimica Polonica 52, no. 3 (August 4, 2005): 647–53. http://dx.doi.org/10.18388/abp.2005_3425.
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Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.
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McCallum, Matthew, Gary S. Shaw, and Carole Creuzenet. "Characterization of the dehydratase WcbK and the reductase WcaG involved in GDP-6-deoxy-manno-heptose biosynthesis in Campylobacter jejuni." Biochemical Journal 439, no. 2 (September 28, 2011): 235–48. http://dx.doi.org/10.1042/bj20110890.
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The capsule of Campylobacter jejuni strain 81-176 comprises the unusual 6-deoxy-α-D-altro-heptose, whose biosynthesis and function are not known. In the present study, we characterized enzymes of the capsular cluster, WcbK and WcaG, to determine their role in 6-deoxy-altro-heptose synthesis. These enzymes are similar to the Yersinia pseudotuberculosis GDP-manno-heptose dehydratase/reductase DmhA/DmhB that we characterized previously. Capillary electrophoresis and MS analyses showed that WcbK is a GDP-manno-heptose dehydratase whose product can be reduced by WcaG, and that WcbK/WcaG can use the substrate GDP-mannose, although with lower efficiency than heptose. Comparison of kinetic parameters for WcbK and DmhA indicated that the relaxed substrate specificity of WcbK comes at the expense of catalytic performance on GDP-manno-heptose. Moreover, although WcbK/WcaG and DmhA/DmhB are involved in altro- versus manno-heptose synthesis respectively, the enzymes can be used interchangeably in mixed reactions. NMR spectroscopy analyses indicated conservation of the sugar manno configuration during catalysis by WcbK/WcaG. Therefore additional capsular enzymes may perform the C3 epimerization necessary to generate 6-deoxy-altro-heptose. Finally, a conserved residue (Thr187 in WcbK) potentially involved in substrate specificity was identified by structural modelling of mannose and heptose dehydratases. Site-directed mutagenesis and kinetic analyses demonstrated its importance for enzymatic activity on heptose and mannose substrates.
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SUNDARAM, Appavu K., Lee PITTS, Kamilah MUHAMMAD, Jing WU, Michael BETENBAUGH, Ronald W. WOODARD, and Willie F. VANN. "Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni." Biochemical Journal 383, no. 1 (September 24, 2004): 83–89. http://dx.doi.org/10.1042/bj20040218.
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Escherichia coli NeuNAc (N-acetylneuraminic acid) synthase catalyses the condensation of PEP (phosphoenolpyruvate) and ManNAc (N-acetylmannosamine) to form NeuNAc and is encoded by the neuB gene. Campylobacter jejuni has three neuB genes, one of which is very similar to the E. coli neuB gene. We have characterized the C. jejuni neuraminic acid synthase with respect to acylamino sugar specificity and stereochemistry of the PEP condensation. We determined the specificity of C. jejuni NeuNAc synthase for N-acetylmannosamine, N-butanoylmannosamine, N-propionoylmannosamine and N-pentanoylmannosamine. We find that, although this enzyme exhibits similar Km values for N-acylmannosamine molecules with different N-acyl groups, the kcat/Km values decreased with increasing chain length. NeuNAc synthase is a member of a PEP-utilizing family of enzymes that form oxo acids from PEP and a monosaccharide. This family includes KDO 8-P (2-keto-3-deoxy-D-manno-octulosonate 8-phosphate) synthase and DAH 7-P (2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate) synthase. Both enzymes catalyse the condensation of the re face of the aldehyde group of the monosaccharide with the si face of the PEP molecule. The C. jejuni NeuNAc synthase catalysed the condensation of Z- and E-[3-2H]PEP with ManNAc, yielding (3S)-3-deutero-NeuNAc and (3R)-3-deutero-NeuNAc respectively. The condensation of Z-[3-F]PEP and ManNAc yielded (3S)-3-fluoro-NeuNAc. Results of our studies suggest that the C. jejuni NeuNAc synthase, similar to KDO 8-P synthase and DAH 7-P synthase, catalyses the condensation of the si face of PEP with the aldehyde sugar. The present study is the first stereochemical analysis of the reaction catalysed by a bacterial NeuNAc synthase.
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Rawls, Katherine S., Shalane K. Yacovone, and Julie A. Maupin-Furlow. "GlpR Represses Fructose and Glucose Metabolic Enzymes at the Level of Transcription in the Haloarchaeon Haloferax volcanii." Journal of Bacteriology 192, no. 23 (October 8, 2010): 6251–60. http://dx.doi.org/10.1128/jb.00827-10.
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ABSTRACT In this study, a DeoR/GlpR-type transcription factor was investigated for its potential role as a global regulator of sugar metabolism in haloarchaea, using Haloferax volcanii as a model organism. Common to a number of haloarchaea and Gram-positive bacterial species, the encoding glpR gene was chromosomally linked with genes of sugar metabolism. In H. volcanii, glpR was cotranscribed with the downstream phosphofructokinase (PFK; pfkB) gene, and the transcript levels of this glpR-pfkB operon were 10- to 20-fold higher when cells were grown on fructose or glucose than when they were grown on glycerol alone. GlpR was required for repression on glycerol based on significant increases in the levels of PFK (pfkB) transcript and enzyme activity detected upon deletion of glpR from the genome. Deletion of glpR also resulted in significant increases in both the activity and the transcript (kdgK1) levels of 2-keto-3-deoxy-d-gluconate kinase (KDGK), a key enzyme of haloarchaeal glucose metabolism, when cells were grown on glycerol, compared to the levels obtained for media with glucose. Promoter fusions to a β-galactosidase bgaH reporter revealed that transcription of glpR-pfkB and kdgK1 was modulated by carbon source and GlpR, consistent with quantitative reverse transcription-PCR (qRT-PCR) and enzyme activity assays. The results presented here provide genetic and biochemical evidence that GlpR controls both fructose and glucose metabolic enzymes through transcriptional repression of the glpR-pfkB operon and kdgK1 during growth on glycerol.
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Romo, Anthony J., and Hung-wen Liu. "Mechanisms and structures of vitamin B6-dependent enzymes involved in deoxy sugar biosynthesis." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1814, no. 11 (November 2011): 1534–47. http://dx.doi.org/10.1016/j.bbapap.2011.02.003.
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Zepeda, S., O. Monasterio, and T. Ureta. "NADP+-dependent d-xylose dehydrogenase from pig liver. Purification and properties." Biochemical Journal 266, no. 3 (March 15, 1990): 637–44. http://dx.doi.org/10.1042/bj2660637.
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An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.
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Rho, Jung-hyun, Damian P. Wright, David L. Christie, Keith Clinch, Richard H. Furneaux, and Anthony M. Roberton. "A Novel Mechanism for Desulfation of Mucin: Identification and Cloning of a Mucin-Desulfating Glycosidase (Sulfoglycosidase) from Prevotella Strain RS2." Journal of Bacteriology 187, no. 5 (March 1, 2005): 1543–51. http://dx.doi.org/10.1128/jb.187.5.1543-1551.2005.
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ABSTRACT A novel enzyme which may be important in mucin degradation has been discovered in the mucin-utilizing anaerobe Prevotella strain RS2. This enzyme cleaves terminal 2-acetamido-2-deoxy-β-d-glucopyranoside 6-sulfate (6-SO3-GlcNAc) residues from sulfomucin and from the model substrate 4-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside 6-sodium sulfate. The existence of this mucin-desulfating glycosidase (sulfoglycosidase) suggests an alternative mechanism by which this bacterium may desulfate sulfomucins, by glycosidic removal of a sulfated sugar from mucin oligosaccharide chains. Previously, mucin desulfation was thought to take place by the action of a specific desulfating enzyme, which then allowed glycosidases to remove desulfated sugar. Sulfate removal from sulfomucins is thought to be a rate-limiting step in mucin degradation by bacteria in the regions of the digestive tract with a significant bacterial flora. The sulfoglycosidase was induced by growth of the Prevotella strain on mucin and was purified 284-fold from periplasmic extracts. Tryptic digestion and sequencing of peptides from the 100-kDa protein enabled the sulfoglycosidase gene to be cloned and sequenced. Active recombinant enzyme was made in an Escherichia coli expression system. The sulfoglycosidase shows sequence similarity to hexosaminidases. The only other enzyme that has been shown to remove 6-SO3-GlcNAc from glycoside substrates is the human lysosomal enzyme β-N-acetylhexosaminidase A, point mutations in which cause the inheritable, lysosomal storage disorder Tay-Sachs disease. The human enzyme removes GlcNAc from glycoside substrates also, in contrast to the Prevotella enzyme, which acts on a nonsulfated substrate at a rate that is only 1% of the rate observed with a sulfated substrate.
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Zemek, Jiří, and Štefan Kučár. "Biosynthesis of sucrose and its deoxy derivatives." Collection of Czechoslovak Chemical Communications 53, no. 1 (1988): 173–80. http://dx.doi.org/10.1135/cccc19880173.
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The biosynthesis of sucrose (β-D-fructofuranosyl-α-D-glucopyranoside) was studied in the reaction catalyzed by partially purified sucrose syntheses (UDP-D-glucose: D-fructose 2glucosyltransferase) isolated from pea seedlings, bean seedlings and sugar beet roots using UDP-D-glucose and its deoxyglucosyl derivatives as donors and D-fructose and its deoxy-analogues as acceptors. It was found that none of the hydroxyl groups either of the reaction donor or the acceptor is essential for the substrate properties in the transglycosylation reaction. The affinity of these plant enzymes and the rate of hexose incorporation into sucrose decreases however in the following sequence: sucrose, 2G-deoxysucrose, 6G-deoxysucrose, 4G-deoxysucrose, and 3G-deoxysucrose for the donor and sucrose, 6F-deoxysucrose, 1F-deoxysucrose, 4F-deoxysucrose, and 3F-deoxysucrose for the acceptor.
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Grootaert, Hendrik, Linde Van Landuyt, Paco Hulpiau, and Nico Callewaert. "Functional exploration of the GH29 fucosidase family." Glycobiology 30, no. 9 (March 9, 2020): 735–45. http://dx.doi.org/10.1093/glycob/cwaa023.
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Abstract The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family’s large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.
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Gokhale, Uday B., Ole Hindsgaul, and Monica M. Palcic. "Chemical synthesis of GDP-fucose analogs and their utilization by the Lewis *A(1 → 4) fucosyltransferase." Canadian Journal of Chemistry 68, no. 7 (July 1, 1990): 1063–71. http://dx.doi.org/10.1139/v90-165.
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Chemical syntheses are reported for GDP-fucose (5), GDP-3-deoxy-fucose (6), and GDP-arabinose (7), the demethyl analog of 5. All three sugar nucleotides were found to act as donor substrates for an α(1 → 4) fucosyltransferase isolated from human milk when *BDGal(1 → 3)*BDGlcNAc-O(CH2)8COOMe (1) was used as the acceptor. The rate of transfer of sugar residues to 1 was measured using a coupled spectrophotometric assay and was found to be 100% (5), 2.3% (6), and 5.9% (7). The product Lea-active oligosaccharide analogs were identified by both an enzyme-linked immunosorbent assay (ELISA) and by 1H NMR spectroscopy. Keywords: glycosyltransferase, oligosaccharide synthesis, sugar-nucleotide analog, ELISA assay, fucosyltransferase.
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Tang, Gaoyan, and Keith P. Mintz. "Glycosylation of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans Is Dependent upon the Lipopolysaccharide Biosynthetic Pathway." Journal of Bacteriology 192, no. 5 (January 8, 2010): 1395–404. http://dx.doi.org/10.1128/jb.01453-09.
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ABSTRACT The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.
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Sutter, Jan-Moritz, Julia-Beate Tästensen, Ulrike Johnsen, Jörg Soppa, and Peter Schönheit. "Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase." Journal of Bacteriology 198, no. 16 (June 13, 2016): 2251–62. http://dx.doi.org/10.1128/jb.00286-16.
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ABSTRACTThe halophilic archaeonHaloferax volcaniihas been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified fromH. volcanii, and the encoding gene,gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgadmutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for thein vivooperation of the spED pathway for glucose degradation inH. volcanii.IMPORTANCEThe work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeonHaloferax volcanii. The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for thein vivooperation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domainArchaea.
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Wang, Ying, Yanli Xu, Andrei V. Perepelov, Yuanyuan Qi, Yuriy A. Knirel, Lei Wang, and Lu Feng. "Biochemical Characterization of dTDP-d-Qui4N and dTDP-d-Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7." Journal of Bacteriology 189, no. 23 (September 28, 2007): 8626–35. http://dx.doi.org/10.1128/jb.00777-07.
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ABSTRACT O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-d-glucose (d-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-d-glucose (d-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-d-Qui4N and dTDP-d-Qui4NAc (the nucleotide-activated precursors of d-Qui4NGlyAc and d-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-d-Qui4N is synthesized from α-d-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-d-Qui4N into dTDP-d-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioAD7) and E. coli O7 (VioAO7) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-d-Qui4N and dTDP-d-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.
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Rupprath, Carsten, Maren Kopp, Dennis Hirtz, Rolf Müller, and Lothar Elling. "An Enzyme Module System forin situ Regeneration of Deoxythymidine 5′-Diphosphate (dTDP)-Activated Deoxy Sugars." Advanced Synthesis & Catalysis 349, no. 8-9 (June 4, 2007): 1489–96. http://dx.doi.org/10.1002/adsc.200700058.
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Parakkottil Chothi, Madhu, Garry A. Duncan, Andrea Armirotti, Chantal Abergel, James R. Gurnon, James L. Van Etten, Cinzia Bernardi, Gianluca Damonte, and Michela Tonetti. "Identification of an l-Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses." Journal of Virology 84, no. 17 (June 10, 2010): 8829–38. http://dx.doi.org/10.1128/jvi.00770-10.
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ABSTRACT Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar l-rhamnose: two UDP-d-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-d-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection.
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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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Klaffke, Werner. "ChemInform Abstract: The Enzyme DTDP-glucose-4,6-dehydratase as a Tool for the Synthesis of Deoxy Sugars." ChemInform 31, no. 44 (October 31, 2000): no. http://dx.doi.org/10.1002/chin.200044279.
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Kubler-Kielb, Joanna, Bruce Coxon, and Rachel Schneerson. "Chemical Structure, Conjugation, and Cross-Reactivity of Bacillus pumilus Sh18 Cell Wall Polysaccharide." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6891–901. http://dx.doi.org/10.1128/jb.186.20.6891-6901.2004.
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ABSTRACT Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-α-galactopyranose (13%) and 2-acetamido-2-deoxy-α-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of →3-O-(2-acetamido-2-deoxy-β-glucopyranosyl)-1→4-ribitol-1-OPO3→. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, including H. influenzae types a and b and strains of Staphylococcus aureus and Staphylococcus epidermidis.
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KNEIDINGER, Bernd, Suzon LAROCQUE, Jean-Robert BRISSON, Nicolas CADOTTE, and Joseph S. LAM. "Biosynthesis of 2-acetamido-2,6-dideoxy-l-hexoses in bacteria follows a pattern distinct from those of the pathways of 6-deoxy-l-hexoses." Biochemical Journal 371, no. 3 (May 1, 2003): 989–95. http://dx.doi.org/10.1042/bj20030099.
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6-Deoxy-l-hexoses have been shown to be synthesized from dTDP-d-glucose or GDP-d-mannose so that the gluco/galacto-configuration is converted into the manno/talo-configuration, and manno/talo is switched to gluco/galacto. Our laboratory has been investigating the biosynthesis of 2-acetamido-2,6-dideoxy-l-hexoses in both Gram-positive and Gram-negative bacteria, and in a recent paper we described the biosynthesis of the talo (pneumosamine) and galacto (fucosamine) derivatives from UDP-d-N-acetylglucosamine a 2-acetamido sugar [Kneidinger, O'Riordan, Li, Brisson, Lee and Lam (2003) J. Biol. Chem. 278, 3615–3627]. In the present study, we undertake the task to test the hypothesis that UDP-d-N-acetylglucosamine is the common precursor for the production of 2-acetamido-2,6-dideoxy-l-hexoses in the gluco-, galacto-, manno- and talo-configurations. We present data to reveal the steps for the biosynthesis of the gluco (quinovosamine)- and manno (rhamnosamine)-configured compounds. The corresponding enzymes WbvB, WbvR and WbvD from Vibrio cholerae serotype O37 have been overexpressed and purified to near homogeneity. The enzymic reactions have been analysed by capillary electrophoresis and NMR spectroscopy. Our data have revealed a general feature of reaction cascades due to the three enzymes. First, UDP-d-N-acetylglucosamine is catalysed by the multi-functional enzyme WbvB, whereby dehydration occurs at C-4, C-6 and epimerization at C-5, C-3 to produce UDP-2-acetamido-2,6-dideoxy-l-lyxo-4-hexulose. Secondly, this intermediate is converted by the C-4 reductase, WbvR, in a stereospecific reaction to yield UDP-2-acetamido-l-rhamnose. Thirdly, UDP-2-acetamido-l-rhamnose is epimerized at C-2 to UDP-2-acetamido-l-quinovose by WbvD. Interestingly, WbvD is also an orthologue of WbjD, but not vice versa. Incubation of purified WbvD with UDP-2-acetamido-2,6-dideoxy-l-talose and analysing the reaction products by capillary electrophoresis revealed the same product peak as when WbjD was used. This sugar nucleotide is a specific substrate for WbjD and is a C-4 epimer of UDP-2-acetamido-l-rhamnose.
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Wagstaff, Ben A., Azul Zorzoli, and Helge C. Dorfmueller. "NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature." Biochemical Journal 478, no. 4 (February 18, 2021): 685–701. http://dx.doi.org/10.1042/bcj20200505.
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Rhamnose is an important 6-deoxy sugar present in many natural products, glycoproteins, and structural polysaccharides. Whilst predominantly found as the l-enantiomer, instances of d-rhamnose are also found in nature, particularly in the Pseudomonads bacteria. Interestingly, rhamnose is notably absent from humans and other animals, which poses unique opportunities for drug discovery targeted towards rhamnose utilizing enzymes from pathogenic bacteria. Whilst the biosynthesis of nucleotide-activated rhamnose (NDP-rhamnose) is well studied, the study of rhamnosyltransferases that synthesize rhamnose-containing glycoconjugates is the current focus amongst the scientific community. In this review, we describe where rhamnose has been found in nature, as well as what is known about TDP-β-l-rhamnose, UDP-β-l-rhamnose, and GDP-α-d-rhamnose biosynthesis. We then focus on examples of rhamnosyltransferases that have been characterized using both in vivo and in vitro approaches from plants and bacteria, highlighting enzymes where 3D structures have been obtained. The ongoing study of rhamnose and rhamnosyltransferases, in particular in pathogenic organisms, is important to inform future drug discovery projects and vaccine development.
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Richhardt, Janine, Stephanie Bringer, and Michael Bott. "Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a ΔeddΔedaMutant on Mannitol." Applied and Environmental Microbiology 78, no. 19 (July 27, 2012): 6975–86. http://dx.doi.org/10.1128/aem.01166-12.
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ABSTRACTThe obligatory aerobic acetic acid bacteriumGluconobacter oxydans621H oxidizes sugars and sugar alcohols primarily in the periplasm, and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is nonfunctional, and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a Δgndmutant lacking the oxidative PPP and a ΔeddΔedamutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as the carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The ΔeddΔedamutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion ofgnd(6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP also. These Δgnd zwf* mutants (with a mutation in thezwfgene causing inactivation of the glucose-6-phosphate dehydrogenase) were almost totally disabled in fructose catabolism but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression ofgndin the reference strain improved biomass formation in a similar manner as deletion ofeddandeda, further confirming the importance of the PPP for cytoplasmic fructose catabolism.
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Iftikhar, Mehwish, Lin Wang, and Zhijie Fang. "Synthesis of 1-Deoxynojirimycin: Exploration of Optimised Conditions for Reductive Amidation and Separation of Epimers." Journal of Chemical Research 41, no. 8 (August 2017): 460–64. http://dx.doi.org/10.3184/174751917x15000341607489.
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1-Deoxynojirimycin (DNJ), which has importance with respect to sugar processing enzymes, is a synthetic target for chemists. A key step in the synthesis of DNJ is the preparation of 2,3,4,6-tetra- O-benzyl-D-glucono-δ-lactam. By varying reaction parameters such as temperature, solvent and reducing reagent, improvements on previous methods are described. A novel approach for the synthesis of 2,3,4,6-tetra- O-benzyl-5-dehydro-5-deoxo-D-gluconamide has been developed by using PCC as an oxidising agent. Separation of epimers permitted DNJ to be obtained in 85% yield after reduction and hydrogenolysis steps.
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COLLARD, François, Elsa WIAME, Niki BERGANS, Juliette FORTPIED, Didier VERTOMMEN, Florent VANSTAPEL, Ghislain DELPIERRE, and Emile VAN SCHAFTINGEN. "Fructosamine 3-kinase-related protein and deglycation in human erythrocytes." Biochemical Journal 382, no. 1 (August 10, 2004): 137–43. http://dx.doi.org/10.1042/bj20040307.
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Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 °C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9–2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.
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Ovchinnikova, Olga G., Evan Mallette, Akihiko Koizumi, Todd L. Lowary, Matthew S. Kimber, and Chris Whitfield. "Bacterial β-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99)." Proceedings of the National Academy of Sciences 113, no. 22 (May 19, 2016): E3120—E3129. http://dx.doi.org/10.1073/pnas.1603146113.
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Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of β-Kdo residues links “group 2” capsular polysaccharides to (lyso)phosphatidylglycerol. β-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo–dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the β-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype β-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis inRaoultella terrigena. The β-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/β domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities.
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Sarazin, Hervé, Sandrine Prudent, Albert Defoin, and Céline Tarnus. "Evaluation of 6-Deoxy-amino-sugars as Potent Glycosidase Inhibitors. Importance of the CH2 OH(6) Group for Enzyme-Substrate Interaction." ChemistrySelect 2, no. 4 (February 1, 2017): 1484–90. http://dx.doi.org/10.1002/slct.201601961.
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EIS, Christian, and Bernd NIDETZKY. "Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors." Biochemical Journal 363, no. 2 (April 8, 2002): 335–40. http://dx.doi.org/10.1042/bj3630335.
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Trehalose phosphorylase is a component of the α-d-glucopyranosyl α-d-glucopyranoside (α,α-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from α,α-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for α,α-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme—substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (Ki) for deoxy- and deoxyfluoro derivatives of d-glucose show that loss of interactions with the 3-, 4- or 6-OH, but not the reactive 1- and the 2-OH, results in considerably (≥100-fold) weaker affinity for sugar-binding subsite +1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the d-glucose-leaving group/nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed α-retaining phosphorolysis of α-d-glucose-1-fluoride (α-d-Glc-1-F) since the productive bonding capability of the fluoride-leaving group with subsite +1 is minimal. The specificity constant (19M−1·s−1) and catalytic-centre activity (0.1s−1) for the reaction with α-d-Glc-1-F are 0.10- and 0.008-fold the corresponding kinetic parameters for the enzymic reaction with α,α-trehalose. The non-selective-inhibition profile for a series of inactive α-d-glycopyranosyl phosphates shows that the driving force for the binary-complex formation lies mainly in interactions of the enzyme with the phosphate group and suggests that hydrogen bonding with hydroxyl groups at the catalytic site (subsite −1) contributes to catalysis by providing stabilization, which is specific to the transition state. Vanadate, a tight-binding phosphate mimic, inhibits the phosphorolysis of α-d-Glc-1-F by forming a ternary complex whose apparent dissociation constant of 120μM is approx. 160-fold greater than the dissociation constant of the same inhibitor complex with α,α-trehalose.
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Bissonnette, P., H. Gagne, A. Blais, and A. Berteloot. "2-Deoxyglucose transport and metabolism in Caco-2 cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 270, no. 1 (January 1, 1996): G153—G162. http://dx.doi.org/10.1152/ajpgi.1996.270.1.g153.
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We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be attributed to the existence of a predominant pathway with a K(m) of 1.7 +/- 0.2 mM. Finally, analysis of the growth-related expression of DG transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.
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Hao, Jijun, Willie F. Vann, Stephan Hinderlich, and Munirathinam Sundaramoorthy. "Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation." Biochemical Journal 397, no. 1 (June 14, 2006): 195–201. http://dx.doi.org/10.1042/bj20052034.
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The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 Å (1 Å=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.
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Peng, Chang G., and Masad J. Damha. "Probing DNA polymerase activity with stereoisomeric 2′-fluoro-β-D-arabinose (2′F-araNTPs) and 2′-fluoro-β-D-ribose (2′F-rNTPs) nucleoside 5′-triphosphates." Canadian Journal of Chemistry 86, no. 9 (September 1, 2008): 881–91. http://dx.doi.org/10.1139/v08-089.
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2′-Deoxy-2′-fluoro-β-D-ribonucleosides (2′F-rN) and 2′-deoxy-2′-fluoro-β-D-arabinonucleosides (2′F-araN) differ solely in the stereochemistry at the 2′-carbon of the furanose sugar ring. 2′F-rN 5′-triphosphates (2′F-rNTPs) are among the most commonly used sugar-modified nucleoside 5′-triphosphates (NTPs) for in vitro selection; however, the epimeric 2′F-araN 5′-triphosphates (2′F-araNTPs) have only recently been applied to polymerase-directed biosynthesis [C.G. Peng and M.J. Damha. J. Am. Chem. Soc. 129, 5310 (2007)]. The present study describes primer extension assays that compare, for the first time, the incorporation efficiency of the two isomeric NTPs, namely, 2′F-araNTPs or 2′F-rNTPs, by four DNA polymerases [Deep Vent (exo-), 9°Nm, HIV-1 RT, and MMLV-RT]. Under the conditions used, incorporation of 2′F-araTTP proceeded more efficiently relative to 2′F-rUTP, while the incorporation of 2′F-araCTP is comparable or slightly less efficient than that observed with 2′F-rCTP. Interestingly, these preferences were observed for all four of the DNA polymerases tested. Unexpected differences in NTP incorporation were observed for 2′F-rCTP vs. rCTP. Despite their seemingly similar conformation, they behaved striking differently in the in vitro polymerization assays. 2′F-rCTP is a much better substrate than the native counterpart (rCTP), an observation first made with human DNA polymerases [F.C. Richardson, R.D. Kuchta, A. Mazurkiewicz, K.A. Richardson. Biochem. Pharmacol. 59, 1045 (2000)]. In contrast, 2′F-rUTP behaved like rUTP, providing poor yield of full-length products. Taken together, this indicates that 2′F-rCTP is very unusual with regard to enzyme/substrate recognition; an observation that can be exploited for the production of DNA oligomers enriched with both ribose and arabinose modifications. These findings are timely given the significant interest and growing need to develop chemically modified oligonucleotides for therapeutic and diagnostic research. By examining the structure-activity relationship (SAR) of the ribose and arabinose sugar, this study furthers our understanding of how the nature of the 2′ substituent (e.g., α vs. β; F vs. OH) and the heterocyclic base affect NTP selection (specificity) by DNA polymerases.Key words: 2′F-rNTPs, 2′F-araNTPs, DNA polymerases, biosynthesis, modified nucleoside triphosphates.
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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.