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Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

Received 11 April 2002/ Accepted 17 July 2002

Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcß13Gal14Galß14Glc) and isoglobotetraose (GalNAcß13Gal13Galß14Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant ß-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

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