- Exploring the broad nucleotide triphosphate and sugar-1-phosphate specificity of thymidylyltransferase Cps23FL from: Streptococcus pneumonia serotype 23F
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Glucose-1-phosphate thymidylyltransferase (Cps23FL) from Streptococcus pneumonia serotype 23F is the initial enzyme that catalyses the thymidylyl transfer reaction in prokaryotic deoxythymidine diphosphate-l-rhamnose (dTDP-Rha) biosynthetic pathway. In this study, the broad substrate specificity of Cps23FL towards six glucose-1-phosphates and nine nucleoside triphosphates as substrates was systematically explored, eventually providing access to nineteen sugar nucleotide analogs.
- Chen, Zonggang,Gu, Guofeng,Jin, Guoxia,Li, Siqiang,Wang, Hong
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p. 30110 - 30114
(2020/09/07)
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- Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling
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In recent years, the development of the enzymatic bioorthogonal labeling strategy has offered exciting possibilities in the probing of structure-defined glycan epitopes. This strategy takes advantage of relaxed donor specificity and strict acceptor specificity of glycosyltransferases to label target glycan epitopes with bioorthogonal reactive groups carried by unnatural nucleotide sugars in vitro. The subsequent covalent conjugation by bioorthogonal chemical reactions with either fluorescent or affinity tags allows further visualization, quantification, or enrichment of target glycan epitopes. However, the application and development of the enzymatic labeling strategy have been hindered due to the limited availability of unnatural nucleotide sugars. Herein, a platform that combines chemical synthesis and enzymatic synthesis for the facile preparation of unnatural nucleotide sugars modified with diverse bioorthogonal reactive groups is described. By this platform, a total of 25 UDP-GlcNAc and UDP-GalNAc derivatives, including the most well explored bioorthogonal functional groups, were successfully synthesized. Furthermore, the potential application of these compounds for use in enzymatic bioorthogonal labeling reactions was also evaluated.
- Wen, Liuqing,Gadi, Madhusudhan Reddy,Zheng, Yuan,Gibbons, Christopher,Kondengaden, Shukkoor Muhammed,Zhang, Jiabin,Wang, Peng George
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p. 7659 - 7666
(2018/07/21)
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- Compositions and methods for the transfer of a hexosamine to a modified nucleotide in a nucleic acid
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Nucleic acids comprising β-glucosaminyloxy-5-methylcytosine; compositions, kits and methods of producing the nucleic acids using a glycosyltransferase; and methods of using the nucleic acids are described.
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- Enzymatic synthesis of nucleobase-modified UDP-sugars: Scope and limitations
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Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4″-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4″-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.
- Wagstaff, Ben A.,Rejzek, Martin,Pesnot, Thomas,Tedaldi, Lauren M.,Caputi, Lorenzo,O'Neill, Ellis C.,Benini, Stefano,Wagner, Gerd K.,Field, Robert A.
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- CHEMOENZYMATIC SYNTHESIS OF HEPARIN AND HEPARAN SULFATE ANALOGS
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The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.
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Paragraph 0304
(2014/09/03)
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- Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)
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A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine. The Royal Society of Chemistry 2012.
- Muthana, Musleh M.,Qu, Jingyao,Li, Yanhong,Zhang, Lei,Yu, Hai,Ding, Li,Malekan, Hamed,Chen, Xi
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supporting information; experimental part
p. 2728 - 2730
(2012/04/17)
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- One-pot three-enzyme synthesis of UDP-GlcNAc derivatives
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A Pasteurella multocida N-acetylglucosamine 1-phosphate uridylyltransferase (PmGlmU) was cloned and used efficiently with an N-acetylhexosamine 1-kinase (NahK-ATCC55813) and an inorganic pyrophosphatase (PmPpA) for one-pot three-enzyme synthesis of UDP-GlcNAc derivatives with or without further chemical diversification.
- Chen, Yi,Thon, Vireak,Li, Yanhong,Yu, Hai,Ding, Li,Lau, Kam,Qu, Jingyao,Hie, Liana,Chen, Xi
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supporting information; experimental part
p. 10815 - 10817
(2011/11/04)
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- Exploiting nucleotidylyltransferases to prepare sugar nucleotides
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(Graph Presented) Enzymatic approaches to prepare sugar nucleotides are gaining in importance and offer several advantages over chemical synthesis including high yields and stereospecificity. We report the cloning, expression, and purification of two new wild-type thymidylyltransferases and observed catalysis with a wide variety of substrates. Significant product inhibition was not observed with the enzymes studied over a 24 h period, enabling the efficient preparation of 15 sugar nucleotides, clearly demonstrating the synthetic utility of these biocatalysts.
- Timmons, Shannon C.,Mosher, Roy H.,Knowles, Sheryl A.,Jakeman, David L.
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p. 857 - 860
(2007/10/03)
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- Chemoenzymatic synthesis of thio-nod factor intermediates - Enzymatic transfer of glucosamine on thiochitobiose derivatives
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The chemoenzymatic syntheses of thioanalogues of nodulation factors in which the nonreducing end glucosamine residue is available for the introduction of the fatty acid moiety at the free NH2 group are reported. We are describing the chemical s
- Morais, Latino Loureiro,Yuasa, Hideya,Bennis, Khalil,Ripoche, Isabelle,Auzanneau, France-Isabelle
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p. 587 - 596
(2007/10/03)
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- One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein
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Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies. Copyright
- Mizanur, Rahman M.,Jaipuri, Firoz A.,Pohl, Nicola L.
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p. 836 - 837
(2007/10/03)
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- Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides
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Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugar nucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization mass spectrometry (ESI-MS)-based assay its unusual tolerance of heat, pH, and sugar substrates. The metalion-dependent enzyme from Pyrococcus furiosus DSM 3638 showed a relatively high degree of acceptance of glucose-1-phosphate (Glc1P), mannose-1-phosphate (Man1P), galactose-1-phosphate (Gal1P), fucose-1-phosphate, glucosamine-1-phosphate, galactosamine-1-phosphate, and N-acetylglucosamine-1-phosphate with uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelis constants for Glc1P, Man1P, and Gal1P are 13.0 ± 0.7, 15 ± 1, and 22 ± 2 μM, respectively, with corresponding turnover numbers of 2.08, 1.65, and 1.32 s-1, respectively. An initial velocity study indicated an ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlight the potential of archaea as a source of new enzymes for synthesis.
- Mizanur, Rahman M.,Zea, Corbin J.,Pohl, Nicola L.
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p. 15993 - 15998
(2007/10/03)
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- Active-site engineering of nucleotidylyltransferases and general enzymatic methods for the synthesis of natural and "unnatural" UDP- and TDP-nucleotide sugars
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The present invention provides mutant nucleotidylyl-transferases, such as Ep, having altered substrate specificity; methods for their production; and methods of producing nucleotide sugars, which utilize these nucleotidylyl-transferases. The present invention also provides methods of synthesizing desired nucleotide sugars using natural and/or modified Ep or other nucleotidyltransferases; and nucleotide sugars sythesized by the present methods. The present invention further provides new glycosyl phosphates, and methods for making them.
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