Practical enzyme-based syntheses of uridine 5'-diphosphogalactose and uridine 5'-diphospho-N-acetylgalactosamine on a gram scale

Heidlas, Jürgen; Lees, Watson J.; Whitesides, George M.

doi:10.1021/jo00027a029

Cited by 65 publications

(26 citation statements)

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“…Several methods have been proposed for regeneration of the NDP-sugar, such as the use of UDPglucose pyrophosphorylase and pyrophosphatase to recycle UDP-glucose (Heidlas et al, 1992;Ichikawa et al, 1992;Wong et al, 1992). However, the cost of these generation systems is still considered to be high for general large-scale applications.…”

Section: Discussionmentioning

confidence: 99%

Arabidopsisglycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Lim

Ashford²,

Hou³

et al. 2004

Biotech & Bioengineering

242

185

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Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.

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Section: Discussionmentioning

confidence: 99%

Arabidopsisglycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Lim

Ashford²,

Hou³

et al. 2004

Biotech & Bioengineering

242

185

View full text Add to dashboard Cite

show abstract

“…2). By successive one pot enzymatic reactions, 9-[U- 13 C 6 ]Gal was phosphorylated, 16) converted to UDP-[U- 13 C 6 ]Gal, 17), 18) and then attached onto the non-reducing terminal N-acetylglucosamine (GlcNAc) residue of 4a and 5a, giving rise to 6b and 6c, respectively. Three isotopomers of the digalactosyl PA῍oli-gosaccharide, 6a, 6b, and 6c were obtained and these samples were subjected to MALDI-QIT-TOF-MS measurements using recrystallized 2,5-dihydroxybenzoic acid as matrix.…”

Section: Methodsmentioning

confidence: 99%

Discrimination of Isomeric Fragment Ions Observed in Tandem Mass Spectra of Biantennary Oligosaccharides by Use of Selective Isotope Labeling

Kato

Yamaguchi

Takahashi

et al. 2004

J. Mass Spectrom. Soc. Jpn.

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“…NMR spectra were recorded in D 2 O and are in agreement with reported data. [10] Electrospray ionization mass spectrometric (ESI-MS) analysis identified a peak at m/z = 565.0471.…”

Section: Synthesis Of Udp-galmentioning

confidence: 99%

“…[9] Previously, Whitesides and coworkers used commercial enzymes to create a regeneration cycle for the synthesis of UDP-Gal based on the Leloir pathway. [10] Moreover, Ozaki and co-workers established an in vivo production system of UDPGal by combining Escherichia coli and Corynebacterium ammoniagenes. [11] Wang and co-workers developed a non-covalently immobilized multi-enzyme system, called superbeads, for the in vitro synthesis of UDP-Gal using an intermediate regeneration system.…”

Section: Introductionmentioning

confidence: 99%

Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

Chien

Liang

et al. 2012

Adv Synth Catal

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In this study, we describe the direct synthesis of uridine 5'-diphosphate galactose (UDP-Gal) by a wild-type bacterial thymidylyltransferase (RmlA), which is used to synthesize thymidine 5'-diphosphate glucose (TDP-glucose) in nature. By using magnesium (Mg 2+ ) as a cofactor and a reaction temperature of 55 8C, a one hundred milligram-scale synthesis of UDP-Gal was achieved by RmlA. In addition, RmlA was site-specifically and covalently immobilized on magnetic nanoparticles (MNPa) The resulting RmlA-MNP complex retained almost 95% of its activity after reuse in ten consecutive enzyme assays. Furthermore, b-1,4-galactosyltransferase (GalT) from Neisseria meningitides was successfully overexpressed and purified by using an intein-mediated protein expression system. GalT was relatively stable at 25 8C, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesize N-acetyllactosamine (LacNAc) using RmlA and GalT in a sequential addition of enzyme and adjustment of thereaction temperature. These results demonstrate the potential applications of bacterial RmlA in carbohydrate synthesis.

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Practical enzyme-based syntheses of uridine 5'-diphosphogalactose and uridine 5'-diphospho-N-acetylgalactosamine on a gram scale

Cited by 65 publications

References 0 publications

Arabidopsisglycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Arabidopsisglycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides

Discrimination of Isomeric Fragment Ions Observed in Tandem Mass Spectra of Biantennary Oligosaccharides by Use of Selective Isotope Labeling

Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

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