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.
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.
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Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
van Die
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Norberg
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van den Eijnden
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004187:Disaccharides / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004187:Disaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases