Donor substrate regeneration for efficient synthesis of globotetraose and isoglobotetraose.
Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4Glc) and isoglobotetraose (GalNAcbeta1-->3Galalpha1-->3Galbeta1-->4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant beta-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.
Shao
Jun
J
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Zhang
Jianbo
J
Kowal
Przemyslaw
P
Wang
Peng George
PG
D001426:Bacterial Proteins; D005915:Globosides / Q000138:chemical synthesis* / Q000737:chemistry; D006193:Haemophilus influenzae / Q000201:enzymology*; D008968:Molecular Conformation; D017350:N-Acetylgalactosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005915:Globosides; C469335:N-acetylgalactosaminyl-1-3-galactopyranosyl-1-3-galactopyranosyl-1-4-glucopyranose; D009844:Oligosaccharides; C035968:globotetraose; C462020:LgtD protein, Haemophilus influenzae; D017350:N-Acetylgalactosaminyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase