Flexible enzymatic and chemo-enzymatic approaches to a broad range of uridine-diphospho-sugars

Errey, James C.; Mukhopadhyay, Balaram; Kartha, K. P. Ravindranathan; Field, Robert A.

doi:10.1039/b410184g

Cited by 64 publications

(94 citation statements)

References 15 publications

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“…UDP-Galf was prepared from synthetic Galf-1-phosphate (38) using galactose-1-phosphate uridyltransferase and UDPglucose pyrophosphorylase as described by Errey et al (39). All stock solutions were prepared by dissolving the appropriate quantity of UDP-sugar in 100 mM potassium phosphate (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Poulin

Nothaft

Hug

et al. 2010

Journal of Biological Chemistry

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UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-D-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.In nature, hexose sugars are found predominantly in the thermodynamically favored pyranose ring form; however, hexose sugars in the furanose ring form are found in bacteria, fungi, and parasites (1, 2). For example, D-galactofuranose (Galf) 5 is a component in many microbial cell surface oligosaccharides (3,

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Section: Methodsmentioning

confidence: 99%

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Poulin

Nothaft

Hug

et al. 2010

Journal of Biological Chemistry

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“…ure 1). [16] We recently examined the ability of a wild-type recombinant glucopyranosyl 1-phosphate thymidylyltransferase to accept furanosyl 1-phosphates 7-11 as substrates ( Figure 1) and were able to show the stereoselective formation of new dTDP 1,2-cis-furanoses with potential biological properties. [17] Therefore, to the best of our knowledge, there is currently no efficient and versatile chemoenzymatic method to synthesize UDP-1,2-cis-furanoses 2-6, analogues of 1α.…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, enzymatic syntheses of 1α have also been reported. [15,16] The first method is based on the use of a UDP-Galp mutase, but synthetic applications are limited by the equilibrium constant of the reaction, which favours the more thermodynamically stable pyranose form. [15] The second paper describes a galactose-1-phosphate uridylyltransferase (Gal-1-PUT) with broad substrate specificity, the reaction being driven to the side of the formation of 1α thanks to the use of a two-enzyme recycling system (Fig-Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Stereoselective Chemoenzymatic Synthesis of UDP‐1,2‐cis‐furanoses from α,β‐Furanosyl 1‐Phosphates

Peltier¹,

Guégan²,

Daniellou³

et al. 2008

Eur J Org Chem

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The biosynthesis of furanosyl‐containing glycoconjugates is poorly described, mainly due to the lack of UDP‐furanoses. Here we present our effort to synthesize rare nucleotide‐sugars with the aid of a multiple‐enzyme system, notably including galactose‐1‐phosphate uridylyltransferase. Firstly, STD‐NMR techniques were used to probe the broad substrate specificity of this particular enzyme. The chemical synthesis of the needed furanosyl 1‐phosphate starting materials was then performed with unprotected thioimidoyl donors. This led to the first stereoselective chemoenzymatic syntheses of UDP‐β‐L‐arabinofuranose, UDP‐α‐D‐fucofuranose and UDP‐α‐D‐6F‐galactofuranose from starting mixtures of sugar‐phosphates.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

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“…Analytes were eluted using mobile phase A: formic acid 0.3% brought to pH 9.0 with ammonia and mobile phase B: acetonitrile using the following multistep gradient at a flow rate 80 µL/min: 0 min: 2% B; 20 min: 15% B; 26 min: 50% B; 27 min: 90% B; 30 min: 90% B; 31 min: 2% B; 50 min: 2% B. Available sugar nucleotide standards (10 µM) were injected (5 µL) to determine retention time (Table S2) [37] and UDP-β-L-Arap [38] were prepared as previously described. Although between runs there were significant differences in absolute retention times of standards, relative retentions were reasonably reproducible (Table S2).…”

Section: Sugar Nucleotide Profilingmentioning

confidence: 99%

Exploring the Glycans of <em>Euglena gracilis</em>

O’Neill¹,

Kuhaudomlarp²,

Rejzek³

et al. 2017

Preprint

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Abstract:Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin-and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.

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Flexible enzymatic and chemo-enzymatic approaches to a broad range of uridine-diphospho-sugars

Abstract: Enzymatic and chemo-enzymatic approaches provide straightforward access to uridine diphospho sugars irrespective of the relative and absolute configuration of the sugar, its ring size and substitution pattern.

Cited by 64 publications

References 15 publications

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Stereoselective Chemoenzymatic Synthesis of UDP‐1,2‐cis‐furanoses from α,β‐Furanosyl 1‐Phosphates

Exploring the Glycans of <em>Euglena gracilis</em>

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