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Biophysical basis for the chain termination in the enacyloxin polyketide synthase

烯酰氧聚酮合酶链终止的生物物理学基础

基本信息

批准号：
BB/L022761/1
负责人：
Józef Lewandowski
金额：
$ 52.78万
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL022761%2F1
关键词：
Biophysical basis chain termination enacyloxin

项目摘要

We face many health related challenges in our everyday life. One of the major challenges is the emergence of multidrug resistant bacteria, which progressively render our arsenal of antibiotics ineffective against them. This rapidly growing problem may eventually lead to a situation where even the smallest infections, e.g. from a scratch, can become lethal as it was common in the pre-antibiotic era. In order to avoid such a situation, there is an urgent need to develop new antibiotics that are effective against disease-causing microorganisms with resistance to the currently available drugs. Enacyloxin IIa has been shown to possess antibacterial activity against the multidrug resistant bacteria Acinetobacter baumannii that is an increasing cause for hospital-acquired infections around the world. Enacyloxin is not stable enough for direct clinical applications but with a number of modifications it could be possibly turned into an effective drug. However, due to its complex structure enacyloxin is difficult to synthesise from scratch. At the same time, the polyketide synthetic biology field has progressed over last 30 years to the point where producing and modifying enacyloxin biosynthetically is a viable alternative.Synthetic biology strives to construct new molecules by exploiting and modifying the biosynthetic machineries available in nature. In particular, polyketide synthases (PKSs) are nature's very large modular enzymatic assembly lines for a wide range of natural products with medicinal properties, ranging from antitumor agents through cholesterol-lowering agents to antibiotics. Polyketide-derived molecules comprise 20% of the top-selling drugs, with the combined worldwide revenues of over £10 billion per year. Due to their modular nature PKSs can be effectively modified to synthesise new compounds. The approach based on mixing and matching components from different assembly lines is very successful with a few hundred new molecules being synthesised to date. Yet, in order to harness these systems for rational production of new compounds, such as enacyloxin analogues, we need to understand the molecular structures and dynamics responsible for specificity and directionality of biosynthesis. In this project we shall obtain such insights about biosynthesis of enacyloxin. To achieve that we propose to study molecular details of enacyloxin PKS and in particular, atomic resolution structures, motions and interactions of the components involved in controlling the crucial step of chain release where two separately assembled molecules are joined together through an ester bond. To obtain the required structural and dynamical insights, we propose employing a combination of highly complementary solution and solid-state magic angle spinning NMR spectroscopies. The proposed approach will enable us, for the first time, to learn how the structure of the proteins evolve on the time scale in the full relevant range from picoseconds to milliseconds. It will also enable us to access direct structural and dynamical information on the large complex of the chain-releasing enzyme and substrate-carrying protein. The solid-state NMR studies on this type of system will be the first of its kind.This project will result in better understanding of enacyloxin biosynthesis and will enable deployment of the studied molecular machinery as a general tool for synthetic biology and synthesis of other compounds. This proposed approach is highly complementary to other structural biology approaches, such as x-ray crystallography and cryo-EM.

我们在日常生活中面临着许多与健康有关的挑战。主要挑战之一是耐多药细菌的出现，这些细菌逐渐使我们的抗生素库对它们无效。这一迅速增长的问题可能最终导致这样一种情况，即即使是最小的感染，例如划伤，也可能致命，因为这在抗生素出现之前的时代很常见。为了避免这种情况，迫切需要开发新的抗生素，以有效对抗对现有药物产生耐药性的致病微生物。已显示对多药耐药细菌鲍曼不动杆菌具有抗菌活性，鲍曼不动杆菌是世界各地医院获得性感染的一个日益增加的原因。对于直接临床应用来说，那昔洛辛还不够稳定，但经过一些修改，它可能会变成一种有效的药物。然而，由于其结构复杂，难以从头合成。与此同时，聚酮合成生物学领域在过去的30年里已经取得了很大的进展，生物合成生产和修饰乙酰胆碱是一种可行的替代方法。合成生物学致力于通过开发和修改自然界中可用的生物合成机制来构建新分子。特别是，聚酮合成酶（pks）是自然界非常大的模块化酶装配线，用于广泛的具有药用特性的天然产物，从抗肿瘤剂到降胆固醇剂再到抗生素。聚酮衍生分子占最畅销药物的20%，全球年收入超过100亿英镑。由于它们的模块化性质，pks可以被有效地修饰以合成新的化合物。这种基于混合和匹配来自不同装配线的成分的方法非常成功，迄今已合成了数百种新分子。然而，为了利用这些系统来合理地生产新化合物，如萘基loxin类似物，我们需要了解生物合成的特异性和方向性的分子结构和动力学。在这个项目中，我们将获得这样的见解对生物合成的纳西洛辛。为了实现这一目标，我们建议研究乙酰氧胺PKS的分子细节，特别是原子分辨率结构、运动和相互作用，这些成分参与控制链释放的关键步骤，其中两个单独组装的分子通过酯键连接在一起。为了获得所需的结构和动力学见解，我们建议采用高度互补的溶液和固态魔角旋转核磁共振光谱的组合。所提出的方法将使我们第一次能够了解蛋白质的结构如何在从皮秒到毫秒的完整相关范围内的时间尺度上进化。这也将使我们能够直接获得链释放酶和底物携带蛋白的大复合体的结构和动力学信息。固体核磁共振对这类体系的研究将是第一次。该项目将使人们更好地了解炔诺酮的生物合成，并使所研究的分子机制成为合成生物学和其他化合物合成的通用工具。这种提出的方法是高度补充其他结构生物学方法，如x射线晶体学和低温电镜。