- Kinetic and NMR spectroscopic study of the chemical stability and reaction pathways of sugar nucleotides
-
The alkaline cleavage of two types of sugar nucleotides has been studied by 1H and 31P NMR in order to obtain information on the stability and decomposition pathways in aqueous solutions under alkaline conditions. The reaction of glucose 1-UDP is straightforward, and products are easy to identify. The results obtained with ribose 5-UDP and ribose 5-phosphate reveal, in contrast, a more complex reaction system than expected, and the identification of individual intermediate species was not possible. Even though definite proof for the mechanisms previously proposed could not be obtained, all the spectroscopic evidence is consistent with them. Results also emphasise the significant effect of conditions, pH, ionic strength, and temperature, on the reactivity under chemical conditions.
- Jaakkola, Juho,Nieminen, Anu,Kivel?, Henri,Korhonen, Heidi,T?htinen, Petri,Mikkola, Satu
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p. 178 - 193
(2020/12/21)
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- Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System
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Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5′- diphospho-α- d-galactose (UDP-Gal) and uridine 5′-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system. (Figure presented.).
- Heinzler, Raphael,Fisch?der, Thomas,Elling, Lothar,Franzreb, Matthias
-
supporting information
p. 4506 - 4516
(2019/08/20)
-
- β-Glucose-1,6-Bisphosphate stabilizes pathological phophomannomutase2 mutants in vitro and represents a lead compound to develop pharmacological chaperones for the most common disorder of glycosylation, PMM2-CDG
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A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, β-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. β-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. β-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.
- Monticelli, Maria,Liguori, Ludovica,Allocca, Mariateresa,Andreotti, Giuseppina,Cubellis, Maria Vittoria
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-
- A mutant of phosphomannomutase1 retains full enzymatic activity, but is not activated by IMP: Possible implications for the disease PMM2-CDG
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The most frequent disorder of glycosylation, PMM2-CDG, is caused by a deficiency of phosphomannomutase activity. In humans two paralogous enzymes exist, both of them require mannose 1,6-bis-phosphate or glucose 1,6-bis-phosphate as activators, but only phospho-mannomutase1 hydrolyzes bis-phosphate hexoses. Mutations in the gene encoding phos-phomannomutase2 are responsible for PMM2-CDG. Although not directly causative of the disease, the role of the paralogous enzyme in the disease should be clarified. Phosphoman-nomutase1 could have a beneficial effect, contributing to mannose 6-phosphate isomerization, or a detrimental effect, hydrolyzing the bis-phosphate hexose activator. A pivotal role in regulating mannose-1phosphate production and ultimately protein glycosylation might be played by inosine monophosphate that enhances the phosphatase activity of phosphoman-nomutase1. In this paper we analyzed human phosphomannomutases by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. We characterized a triple mutant of phospomannomutase1 that retains mutase and phosphatase activity, but is unable to bind inosine monophosphate.
- Citro, Valentina,Cimmaruta, Chiara,Liguori, Ludovica,Viscido, Gaetano,Cubellis, Maria Vittoria,Andreotti, Giuseppina
-
-
- Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases
-
Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-d-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-d-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-d-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.
- Liu, Yuan,Nishimoto, Mamoru,Kitaoka, Motomitsu
-
-
- Efficient chemoenzymatic synthesis of novel galacto-N-biose derivatives and their sialylated forms
-
Galacto-N-biose (GNB) derivatives were efficiently synthesized from galactose derivatives via a one-pot two-enzyme system containing two promiscuous enzymes from Bifidobacterium infantis: a galactokinase (BiGalK) and a d-galactosyl-β1-3-N-acetyl-d-hexosamine phosphorylase (BiGalHexNAcP). Mono-sialyl and di-sialyl galacto-N-biose derivatives were then prepared using a one-pot two-enzyme system containing a CMP-sialic acid synthetase and an α2-3-sialyltransferase or an α2-6-sialyltransferase.
- Li, Lei,Liu, Yonghui,Li, Tiehai,Wang, Wenjun,Yu, Zaikuan,Ma, Cheng,Qu, Jingyao,Zhao, Wei,Chen, Xi,Wang, Peng G.
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supporting information
p. 10310 - 10313
(2015/06/25)
-
- Engineering the specificity of trehalose phosphorylase as a general strategy for the production of glycosyl phosphates
-
A two-step process is reported for the anomeric phosphorylation of galactose, using trehalose phosphorylase as biocatalyst. The monosaccharide enters this process as acceptor but can subsequently be released from the donor side, thanks to the non-reducing nature of the disaccharide intermediate. A key development was the creation of an optimized enzyme variant that displays a strict specificity (99%) for β-galactose 1-phosphate as product. This journal is the Partner Organisations 2014.
- Chen, Chao,Van Der Borght, Jef,De Vreese, Rob,D'Hooghe, Matthias,Soetaert, Wim,Desmet, Tom
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p. 7834 - 7836
(2014/07/08)
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- Purification and characterization of 1,3-β-D-glucan phosphorylase from Ochromonas danica
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1,3-β-D-Glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β- glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25°C-30°C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.
- Yamamoto, Yutaka,Kawashima, Daichi,Hashizume, Ayu,Hisamatsu, Makoto,Isono, Naoto
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p. 1949 - 1954
(2013/10/08)
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- A highly efficient galactokinase from Bifidobacterium infantis with broad substrate specificity
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Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned and over-expressed with a yield of over 80 mg/L cell cultures. The kcat/Km value of recombinant BiGalK toward galactose (164 s-1 mM -1) is 296 times higher than that of GalK from E. coli, indicating that BiGalK is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These features make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and derivatives.
- Li, Lei,Liu, Yonghui,Wang, Wenjun,Cheng, Jiansong,Zhao, Wei,Wang, Peng
-
experimental part
p. 35 - 39
(2012/08/08)
-
- Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase
-
UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH2p) yielding uridine 5′-(β, γ-methylenetriphosphate) (UppCH2p). The f
- Guenther Sillero, Maria Antonia,De Diego, Anabel,Sillero, Antonio
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experimental part
p. 1871 - 1875
(2012/09/22)
-
- Characterization of a bacterial laminaribiose phosphorylase
-
Bacterial laminaribiose phosphorylase (LBPbac) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into α-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBPEug): LBPbac was more specific to laminaribiose than LBPEug. It showed acceptor specificity in reverse phosphorolysis similar to LBPEug. Cloning of the gene encoding LBPbac (lbpA) has revealed that LBPbac is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N0-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of lbpA, suggesting that the role of LBPbac is to utilize laminaribiose generated outside the cell. This role is different from that of LBPEug, which participates in the utilization of paramylon, the intracellular storage 1,3-β-glucan.
- Kitaoka, Motomitsu,Matsuoka, Yasuyuki,Mori, Kiyotaka,Nishimoto, Mamoru,Hayashi, Kiyoshi
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experimental part
p. 343 - 348
(2012/08/08)
-
- Substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4 towards galactose, glucose, and their derivatives
-
Galactokinases (GalKs) have attracted significant research attention for their potential applications in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 presents a remarkably broad substrate range including 14 diverse natural and unnatural sugars. TLC and MS studies revealed that GalKSpe4 had relaxed activity towards galactose derivatives with modifications on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable. More interestingly, GalKSpe4 can phosphorylate l-mannose in moderate yield (43%), while other l-sugars such as l-Gal cannot be recognized by this enzyme. These results are very significant because there is rarely enzyme reported that can phosphorylate such uncommon substrates as l-mannose.
- Zou, Yang,Wang, Wenjun,Cai, Li,Chen, Leilei,Xue, Mengyang,Zhang, Xiaomei,Shen, Jie,Chen, Min
-
body text
p. 3540 - 3543
(2012/07/03)
-
- Pyrimidine nucleotides with 4-alkyloxyimino and terminal tetraphosphate δ-ester modifications as selective agonists of the P2Y4 receptor
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P2Y2 and P2Y4 receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We structurally varied phosphate and uracil moieties in analogues of pyrimidine nucleoside 5′-triphosphates and 5′-tetraphosphate esters. P2Y4 receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N4-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5′-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N4-(Phenylpropoxy)-CTP 16 (MRS4062), Up 4-[1]3′-deoxy-3′-fluoroglucose 34 (MRS2927), and N 4-(phenylethoxy)-CTP 15 exhibit ≤10-fold selectivity for human P2Y4 over P2Y2 and P2Y6 receptors (EC 50 values 23, 62, and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up4 activated P2Y2 but not P2Y 4 receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y2 and P2Y4 receptor models indicated greater steric tolerance of N4-phenylpropoxy group at P2Y4. Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y4 receptor-selective agonists.
- Maruoka, Hiroshi,Jayasekara, M. P. Suresh,Barrett, Matthew O.,Franklin, Derek A.,De Castro, Sonia,Kim, Nathaniel,Costanzi, Stefano,Harden, T. Kendall,Jacobson, Kenneth A.
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scheme or table
p. 4018 - 4033
(2011/08/05)
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- Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4
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Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45 °C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.
- Chen, Min,Chen, Lei-Lei,Zou, Yang,Xue, Mengyang,Liang, Min,Jin, Lan,Guan, Wan-Yi,Shen, Jie,Wang, Wenjun,Wang, Lei,Liu, Jun,Wang, Peng George
-
experimental part
p. 2421 - 2425
(2011/12/15)
-
- The role of the active site residues in human galactokinase: Implications for the mechanisms of GHMP kinases
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Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pKa values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pKa value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C 1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the γ-phosphorus of ATP.
- Megarity, Clare F.,Huang, Meilan,Warnock, Claire,Timson, David J.
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experimental part
p. 120 - 126
(2011/06/25)
-
- Transglucosylation potential of six sucrose phosphorylases toward different classes of acceptors
-
In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes' specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on L-sorbose and L-arabinose, besides the native acceptors D-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineeringwill, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase.
- Aerts, Dirk,Verhaeghe, Tom F.,Roman, Bart I.,Stevens, Christian V.,Desmet, Tom,Soetaert, Wim
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experimental part
p. 1860 - 1867
(2011/12/04)
-
- The binding of β-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors
-
Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(β-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC50 values ranging from 5.7 to 524.3 μM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23 resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO2, F, Cl, Br, OH, OMe, CF3, or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the β-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the β-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.
- Alexacou, Kyra-Melinda,Tenchiu, Alia-Cristina,Chrysina, Evangelia D.,Charavgi, Maria-Despoina,Kostas, Ioannis D.,Zographos, Spyros E.,Oikonomakos, Nikos G.,Leonidas, Demetres D.
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experimental part
p. 7911 - 7922
(2011/02/22)
-
- Efficient synthesis of α-D-glucose-1-phosphate by maltodextrin phosphorylase immobilized on amino-functionalized magnetic nanoparticles
-
α-D-Glucose-1-phosphate (Glc-1-P) is an expensive intermediate in the biosynthesis of nucleotide glucose. This paper describes a biocatalytic system for the efficient synthesis of Glc-1-P from the low cost raw materials: maltodextrin and phosphate at ordinary temperatures. After molecular cloning and the expression of a maltodextrin phosphorylase (MalPase) gene from E. coli (Escherichia coli) K12, the resultant recombinant enzyme was immobilized on amino-functionalized magnetic nanoparticles for recycling and repeated use. Conditions for the biocatalytic reaction were optimized and the immobilized MalPase could be easily recovered and reused over eight cycles in the repeated synthesis of Glc-1-P. After simple purification steps approximately 440 mg of crude product was obtained with a moderate isolation yield of 70.5.
- Dong, Qing,Ouyang, Liming,Liu, Jianwen,Xu, Jianhe
-
experimental part
p. 1227 - 1232
(2010/12/25)
-
- Leishmania UDP-sugar pyrophosphorylase: The missing link in galactose salvage?
-
The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.
- Damerow, Sebastian,Lamerz, Anne-Christin,Haselhorst, Thomas,Fuehring, Jana,Zarnovican, Patricia,von Itsztein, Mark,Routier, Francoise H.
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experimental part
p. 878 - 887
(2010/12/18)
-
- Synthesis of |β-(1→2)-linked oligomannosides
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β-(1→2)-Linked oligomannosides constitute an important class of carbohydrate structures located on the cell surface of several Candida species, including C. albicans. As a result of the immunostimulating properties of such compounds, the upscaling of their synthesis is relevant. In this paper, a highly stereoselective synthesis of |β-(1→2)-linked oligomannosides was performed by further development of and modifications to the methodologies described earlier in the literature. In addition to the synthesis of fully deprotected β-(1→2)-linked mannobiose and mannotriose, some preliminary modifications to the oligosaccharide core, resulting in close analogues with biological potential, are presented. The fully deprotected products form potential targets for screening against C. albicans and may also result in new model structures for vaccine development.
- Polakova, Monika,Roslund, Mattias U.,Ekholm, Filip S.,Saloranta, Tiina,Leino, Reko
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experimental part
p. 870 - 888
(2009/07/17)
-
- Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk
-
A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500mM of LNB was generated in 10-liter of reaction mixture initially containing 660 mM of sucrose and 600mM of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose - hexose-1-phospate uridylyltransferase, UDP-glucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.
- Nishimoto, Mamoru,Kitaoka, Motomitsu
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p. 2101 - 2104
(2008/02/13)
-
- Crystallographic studies on two bioisosteric analogues, N-acetyl-β-D-glucopyranosylamine and N-trifluoroacetyl-β-D- glucopyranosylamine, potent inhibitors of muscle glycogen phosphorylase
-
Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of β-d-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-β-d- glucopyranosylamine (NAG) (Ki = 32 μM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 A resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-β-d- glucopyranosylamine (NFAG), with a Ki = 75 μM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 A resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 A). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the Ki values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.
- Anagnostou, Eleni,Kosmopoulou, Magda N.,Chrysina, Evangelia D.,Leonidas, Demetres D.,Hadjiloi, Theodoros,Tiraidis, Costantinos,Zographos, Spyros E.,Gyoergydeak, Zoltan,Somsak, Laszlo,Docsa, Tibor,Gergely, Pal,Kolisis, Fragiskos N.,Oikonomakos, Nikos G.
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p. 181 - 189
(2007/10/03)
-
- Studies on the substrate specificity of Escherichia coli galactokinase
-
(Martix presented) In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes.
- Yang, Jie,Fu, Xun,Jia, Qiang,Shen, Jie,Biggins, John B.,Jiang, Jiqing,Zhao, Jingjing,Schmidt, Joshua J.,Wang, Peng G.,Thorson, Jon S.
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p. 2223 - 2226
(2007/10/03)
-
- Characterization of trehalose phosphorylase from Bacillus stearothermophilus SK-1 and nucleotide sequence of the corresponding gene
-
A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The Mr of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75°C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a Mr of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.
- Inoue, Yasushi,Ishii, Keiko,Tomita, Tetsuji,Yatake, Tsuneya,Fukui, Fumio
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p. 1835 - 1843
(2007/10/03)
-
- One-step, stereocontrolled synthesis of glycosyl 1-phosphates, uridine- 5'-diphosphogalactose, and uridine-5'-diphosphoglucose from unprotected glycosyl donors
-
The reaction of 2-(1,2-trans-glycopyranosyloxy)-3-methoxypyridines (MOP glycosides) with phosphoric acid leads to the corresponding 1,2-cis-1- phosphates in good yield and excellent stereoselectivity. 1-Phosphate esters of α-D-glucopyranose, α-D-galactopyranose, and 2-azido-2-deoxy-α-D- galactopyranose were thus prepared without recourse to protective groups. In the L-fucose series, the major product was the α-L-fucosyl 1-phosphate. An alternative method that relies on neighboring group participation allowed the preparation of a protected β-L-fucosyl 1-phosphate. Reaction of unprotected β-D-glucopyranosyloxy and β-D-galactopyranosyloxy MOP donors with uridine diphosphoric acid gave UDP-Glc and UDP-Gal with preponderance of the desired α-anomeric configuration.
- Hanessian, Stephen,Lu, Pu-Ping,Ishida, Hideki
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p. 13296 - 13300
(2007/10/03)
-
- Chemo-enzymic Synthesis of Guanosine 5'-Diphosphomannose (GDP-Mannose) and Selected Analogues
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Guanosine 5'-diphosphomannose (GDP-mannose, 1) has been prepared via two routes from α-D-mannose 1-phosphate 5.The non-enzymic route involves coupling of the phosphate 5 to guanosine 5'-monophosphate dibutyl phosphinothioic anhydride (GMP-anhydride 11) to give compound 1 in 40percent yield.The enzymic routes require coupling of compound 5 with guanosine 5'-triphosphate (GTP) catalysed by GDP-mannose pyrophosphorylase (37percent).
- Pallanca, Jane E.,Turner, Nicolas J.
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p. 3017 - 3022
(2007/10/02)
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- Synthesis of galactose-terminated oligosaccharides by use of galactosyltransferase
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Galactosyltransferase catalyzes the galactosylation of oligosaccharides terminated by glucose and by 2-acetamido-2-deoxy glucopyranose, respectively. Variations concerning the acceptor substrate as well as the donor substrate are described.
- Thiem,Wiemann
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p. 141 - 145
(2007/10/02)
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- HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC CHARACTERIZATION OF THE ROLE OF INORGANIC PYROPHOSPHATASE IN REGULATING THE REACTION OF URIDINE 5'-TRIPHOSPHATE WITH GLUCOSE 1-MONOPHOSPHATE
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Inorganic pyrophosphatase (EC 3.6.1.1) drived the reaction of uridine 5'-triphosphate with glucose 1-monophosphate in the direction of uridine 5'-diphosphoglucose formation.
- Yoza, Norimasa,Hirano, Hisanobu,Baba, Yoshinobu,Ohashi, Shigeru
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p. 605 - 610
(2007/10/02)
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- Glycosyl Imidates, 10. - Glycosyl Phosphates from Glycosyl Trichloroacetimidates
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The glycosyl trichloroacetimidates 1-α,β, 8-α, 11-α, 13-α, 15-α, and 17-α directly transfer the glycosyl moiety to phosphoric acid mono- and diesters.The acidity of the phosphoric acid derivatives used was sufficient for the activation of the trichloroacetimidates, therefore an additional acidic catalyst was not required.From the β-imidate 1-β α-glycosyl phosphate and from the α-imidates preferentially β-glycosyl phosphates were obtained.Reaction of dibenzyl phosphate (2a) and cetyl phosphate (2c) with 1-α demonstrated that β/α-anomerization iscatalyzed by traces of acid.The glycosyl phosphates 3a-α, 3a-β - 3c-β, 3f-α, 9a-β, and 9c-β were partly or totally deprotected.The structures of the synthezised compounds were assigned by 1H NMR spectroscopy and by comparison with literature data.
- Schmidt, Richard R.,Stumpp, Michael
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p. 680 - 691
(2007/10/02)
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- SYNTHESIS OF GLYCOSYL PHOSPHATES FROM SUGAR ORTHO ESTERS: FORMATION OF BIS(2,3,4,6-TETRA-O-ACETYL-β-D-GLUCOPYRANOSYL) PHOSPHATE, AND THE EFFECT OF SOLVENTS ON THE SYNTHESIS OF β-D-GLUCOPYRANOSYL PHOSPHATE
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Reaction of exo-3,4,6-tri-O-acetyl-1,2-O-(tert-butyl orthoacetyl)-α-D-glucopyranose with phosphoric acid and phosphorus pentaoxide in oxolane gave 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl phosphate.Treatment of the reaction mixture with aqueous lithium hydroxide gave the expected β-D-glucopyranosyl phosphate.However, neutralization with ammonium hydroxide gave exclusively the phosphoric diester, namely, bis(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) phosphate.Formation of the diester was traced to the phosphorus pentaoxide in the initial reaction-mixture.A study of solvents showed that phosphorylation of the ortho ester proceeds rapidly in a variety of ethers and N,N-dimethylamides.
- Salam, Mohammed A.,Behrman, Edward J.
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