Assembly of chimeric glycosyltransferases for directing biosynthesis of natural products

该项目旨在结合剑桥大学两个已建立的研究团队的努力，解决化学和生物化学领域的一个重大突出问题：如何利用蛋白质工程生成可能具有重要生物活性的新型糖基化化合物。目前临床上使用的很大一部分药物，特别是抗感染药物，如红霉素或万古霉素，是从土壤细菌中分离出来的天然产物或其衍生物。它们所含的不寻常的糖已被证明对生物活动至关重要。天然产物的酶修饰是产生新化合物的一个有吸引力的选择，用不同的糖修饰化合物尤其有吸引力。一类主要的糖基转移酶（GTs），负责将糖从供体核苷酸二磷酸（NDP）糖转移到受体分子的酶，已知由两个结构域组成。一个结合ndp -糖供体，另一个结合受体分子。我们的初步研究表明，通过剪切和粘贴来自不同gt的结构域，可以构建杂交酶，这些酶保持高活性，并且具有它们所衍生的亲本gt的各自供体和受体特异性。这一发现导致了几种新型万古霉素类似物的合成。这些原理验证实验为从超过19,000种已知亲本蛋白中制备大范围的杂交gt铺平了道路，为获取新化合物提供了催化剂工具箱。构建和开发这些杂交酶将需要多种实验方法，从使用纯化酶在试管中合成新化合物到在细菌细胞内针对新天然产物的基因进行遗传操作。该项目的成功将为制药业带来广泛的潜在利益。

This project seeks to combine the efforts of two established research teams in Cambridge to solve a major outstanding problem in chemistry and biochemistry: how to use protein engineering to generate novel glycosylated compounds that may have important biological activities. A significant fraction of drugs currently used in the clinic, especially anti-infectives such as erythromycins or vancomycins, are natural products isolated from soil bacteria, or derivatives of them. The unusual sugars they contain have been shown to be vital for biological activity. Enzymic modification of natural products is an attractive option to generate new compounds and decorating compounds with different sugars is particularly appealing. One major class of glycosyltransferases (GTs), the enzymes responsible for transferring a sugar from the donor nucleotide diphosphate (NDP) sugar to the acceptor molecule, is known to be composed of two domains. One binds the NDP-sugar donor and the other the acceptor molecule. Our preliminary studies have shown that by cutting and pasting domains from different GTs, hybrid enzymes can be constructed that remain highly active and have the respective donor and acceptor specificity of the parent GTs from which they were derived. This finding has led to the synthesis of several novel vancomycin analogues. These proof of principle experiments pave the way for making a wide range of hybrid GTs from over 19,000 known parent proteins to provide a toolbox of catalysts for accessing novel compounds. Constructing and exploiting these hybrid enzymes will require diverse experimental approaches from carrying out the synthesis of the novel compounds in a test tube with the purified enzymes to genetic manipulation of the genes inside the bacterial cells targetting novel natural products. Success in this project would deliver broad potential benefits to the pharmaceutical industry.