- Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina
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Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The km values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.
- Ren, Yan,Perepelov, Andrei V.,Wang, Haiyan,Zhang, Hao,Knirel, Yuriy A.,Wang, Lei,Chen, Wei
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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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- Process for preparing nucleotide inhibitors of glycosyltransferases
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Nucleotide linked 2-deoxy-2-fluoroglycosides are employed as potent competitive inhibitors of glycosyltransferases. More particularly, uridine-5'-diphospho-2-deoxy-2-fluoro-galactose (UDP-2F-Gal), guanidine-5'-diphospho-2-deoxy-2-fluoro-L-fucose (GDP-2F-Fuc), uridine-51-diphospho-2-deoxy-2-fluoro-D-glucose (UDP-2F-Glu), guanosine-5'-diphospho-2-deoxy-2-fluoro-D-mannose (GDP-2F-Man), cytosine-5'-monophospho-2-deoxy-2-fluoro-D-sialic acid, and cytosine-5'-monophospho-2-deoxy-2-KDO may be employed as inhibitors of β-1,4-galactosyltransferase, α-1,3-fucosyltransferase, glucosyltransferases, N-acetylglucosaminyltransferases, (α-mannosyltransferases, α-sialyltransferases, and KDO-transferases, respectively. Synthesis of nucleotide-linked-2-deoxy-2-fluoroglycosides is achieved using either chemoenzymatic or chemical methodologies.
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- Synthesis of guanosine 5′-(β-L-fucopyranosyl)-diphosphate revisited
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It will be demonstrated that a successful synthesis of β-L-fucopyranose-1-phosphate (2), a key intermediate in the preparation of guanosine 5′-(β-L-fucopyranose)-diphosphate (1), strongly depends on the nature of the acyl protecting groups for the non-anomeric hydroxyl functions. Thus, the perbenzoylated, instead of peracetylated, α-L-fucopyranosyl trichloroacetimidate (11) or the corresponding ethyl β-thiofucopyranoside proved to be a convenient starting compound for the preparation of 2. Further, condensation of N,N'-dicyclohexyl-4-morpholinecarboxamidinium guanosine 5′-morpholidophosphate with excess 2 gave the title compound without concomitant formation of bisguanosine-5′-diphosphate (16).
- Heskamp,Broxterman,Van Der Marel,Van Boom
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p. 611 - 622
(2007/10/03)
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- An approach towards the synthesis of 1,2-trans glycosyl phosphates via iodonium ion assisted activation of thioglycosides
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Phosphorylation of benzoylated ethyl 1,2-trans 1-thioglycosides with dibenzyl phosphate in the presence of NIS gave, after removal of all protecting groups, 1,2-trans glycosyl phosphates. The scope of the stereoselective method was demonstrated by the syn
- Veeneman,Broxterman,Van der Marel,Van Boom
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p. 6175 - 6178
(2007/10/02)
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