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
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
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DOI：
10.1093/glycob/9.10.1061
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发表时间：
1999-10-01
期刊：
影响因子：
4.3
通讯作者：
van den Eijnden, DH
中科院分区：
文献类型：
--
作者：
Blixt, O;van Die, I;van den Eijnden, DH
我们在大肠杆菌中高水平表达了脑膜炎奈瑟氏菌lgtA基因，获得了具有高酶活性的编码β-N-乙酰葡糖胺转移酶（简称LgtA），该酶在细菌中参与细菌脂寡糖的乳糖-N-新-四糖结构元件的合成。这种糖基转移酶似乎是不寻常的，因为它显示了广泛的受体特异性对α-和β-半乳糖苷，无论是在结构上相关的N-或O-蛋白，或脂质连接的寡糖。通过一维和二维400 MHz H-1-和C-13-NMR光谱进行的产物分析表明，LgtA催化以β 1->3-键从UDP GlcNAc引入GlcNAc以接受Gal残基。因此，该酶可以表征为UDP-GlcNAc：Gal α β-R β 3-N-乙酰葡糖胺基转移酶。虽然乳糖是高度优选的受体底物，但重组酶也有效地作用于单体和二聚体N-乙酰乳糖胺，揭示了其在酶辅助方法中合成聚乳糖胺聚糖结构中的潜在价值。此外，LgtA显示出对UDP-GalNAc的高供体混杂性，但对其他UDP-糖没有。并且可以以中等速率催化β 1->3-连接的GalNAc引入受体结构中的α-或β-Gal。因此，LgtA显示有望在GlcNAc β 1->3Gal和GalNAc-β 1->3Gal键的制备合成中成为有用的催化剂。
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.