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

We have expressed the Neisseria meningitidis lgtA gene at re high level in Escherichia coli, The encoded beta-N-acetylglucosaminyltransferase, referre d to as LgtA, which in the bacterium is involved in the synthesis of the la cto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransfer ase 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 L gtA catalyzes the introduction of GlcNAc from UDP GlcNAc in a beta 1-->3-li nkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha beta-R beta 3-N-acetylglucosaminyltransferase. Althoug h lactose is a highly preferred acceptor substrate the recombinant enzyme a lso acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures i n enzyme assisted procedures, Furthermore, LgtA shows a high donor promiscu ity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze th e 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.