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 GlcNAcβ1→3Gal and GalNAcβ1-3Gal linkages

We have expressed the Neisseria meningitidis lgtA gene at re 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 H-1- and C-13-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.