Donor substrate promiscuity of bacterial ß1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of ß1-4-galactosyltransferases.

ß1-3-N-Acetylglucosaminyltransferases (ß3GlcNAcTs) and ß1-4-galactosyltransferases (ß4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of ß3GlcNAcTs and ß4GalTs, donor substrate specificity studies of two bacterial ß3GlcNAcTs from Helicobacter pylori (Hpß3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two ß3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three ß4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpß4GalT), and bovine (Bß4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these ß4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcß1-3Galß1-4GlcßProN3 and Galß1-3Galß1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.