Molecular cloning and functional expression of a novel Helicobacter pylori α-1,4 fucosyltransferase
Molecular cloning and functional expression of a novel Helicobacter pylori α-1,4 fucosyltransferase
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DOI：
10.1093/glycob/cwj004
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发表时间：
2005-11-01
期刊：
影响因子：
4.3
通讯作者：
Ernst, B
中科院分区：
文献类型：
--
作者：
Rabbani, S;Miksa, V;Ernst, B
幽门螺杆菌是一种重要的人类病原体，可引起胃和十二指肠溃疡，并与胃癌和淋巴瘤有关。这种微生物合成岩藻糖化低聚糖，主要是Galβ-1，4GlcNAc(II型)血型抗原Lewis X和Y，而一小部分人也表达Gal-beta-1，3GlcNAc(I型)血型抗原Lewis A和B。这些碳水化合物结构已知模仿宿主细胞抗原，允许细菌逃离宿主免疫反应。在这里，我们报道了一个新的幽门螺杆菌α-1，4岩藻糖基转移酶(FUCT)的克隆和特性。与迄今鉴定的家族成员不同，该酶仅显示I型受体底物专一性。该酶由432个氨基酸组成(相对分子质量为50,502 Da)，采用基于聚合酶链式反应的方法进行克隆。它与其他幽门螺杆菌FucT显示出高度的同源性(75%-87%)和相似的结构特征，例如在七聚体重复模式中。动力学研究表明，I型受体底物(Gal)-1，3GlcNAc-Lem(1)有一个非常有效的转移酶(k(CAT)/K-m=229 mm(-1)S(-1))。此外，该酶对非天然I型受体底物类似物具有广泛的耐受性，因此是化学酶法合成Lewis A、sialyl Lewis A及其模拟物的有价值的工具。
Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Gal beta-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Gal beta-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori alpha-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75-87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (k(cat)/K-m = 229 mM(-1)s(-1)) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereof.