High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages

• Published in: Glycobiology (Oxford) 1999-10, Vol.9 (10), p.1061-1071
• Main Authors: Blixt, O, van Die, I, Norberg, T, van den Eijnden, D H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Carbohydrate Conformation; Carbohydrate Sequence; Cloning, Molecular; Disaccharides - biosynthesis; Disaccharides - chemistry; DNA, Bacterial - genetics; Escherichia coli - genetics; Gene Expression; Genes, Bacterial; Kinetics; Magnetic Resonance Spectroscopy; Mammals; Molecular Sequence Data; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Neisseria meningitidis - enzymology; Neisseria meningitidis - genetics; Oligosaccharides - biosynthesis; Oligosaccharides - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Species Specificity; Substrate Specificity
• ISSN: 0959-6658
• DOI: 10.1093/glycob/9.10.1061

We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.