Suppr超能文献

用于抗原性寡糖的程序化化学酶法合成的具有可控特异性的α-转葡糖苷酶的设计

Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides.

作者信息

Champion Elise, André Isabelle, Moulis Claire, Boutet Julien, Descroix Karine, Morel Sandrine, Monsan Pierre, Mulard Laurence A, Remaud-Siméon Magali

机构信息

Universite de Toulouse; INSA, UPS, INP, LISBP, 135 avenue de Rangueil, F-31077 Toulouse, France.

出版信息

J Am Chem Soc. 2009 Jun 3;131(21):7379-89. doi: 10.1021/ja900183h.

Abstract

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.

摘要

结合化学合成,生物催化剂的使用在开辟通往新型分子多样性的道路方面具有巨大潜力。我们报道了体外化学酶促途径,该途径首次利用酶工程来生产复杂的微生物细胞表面寡糖,并突破了糖化学的化学界限。糖基酶被设计用于作用于非天然的方便保护的底物,以产生与程序化化学延伸兼容的中间体。该研究聚焦于模仿福氏志贺氏菌血清型1b和3a的O抗原基序的寡糖合成,这些寡糖可用于开发多价碳水化合物疫苗。一种半理性工程方法成功应用于淀粉蔗糖酶,这是一种使用低成本蔗糖底物作为葡萄糖基供体的转葡萄糖苷酶。主要困难在于保留酶对蔗糖的特异性,同时创造一种新的催化功能,使酶能够区域特异性地将受保护的非天然受体糖基化。构建了一个由133个突变体组成的结构导向文库,从中分离出了几个对α-L-鼠李糖吡喃糖苷具有全新特异性或对烯丙基2-乙酰氨基-2-脱氧-α-D-葡萄糖吡喃糖苷受体具有极大增强特异性的突变体。最佳变体用于合成糖基化构建块。然后将它们转化为与针对两种目标血清型的O抗原寡糖片段的化学延伸兼容的受体和潜在供体。这是首次成功对α-转糖苷酶受体结合位点进行工程改造并产生新特异性的报道。它证明了在糖化学中计划好的化学酶促途径和酶工程的适当组合的潜力。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验