Biophysical basis for the chain termination in the enacyloxin polyketide synthase

我们在日常生活中面临着许多与健康相关的挑战。其中一个主要挑战是多重耐药细菌的出现，这逐渐使我们的抗生素库对它们无效。这个快速增长的问题可能最终导致即使是最小的感染，例如从划痕，也可能变得致命，因为它在前抗生素时代很常见。为了避免这种情况，迫切需要开发新的抗生素，其有效对抗对目前可用药物具有抗性的致病微生物。已证明Enacyloxin IIa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日益增加的原因。Enacloxin对于直接临床应用来说不够稳定，但经过一些修饰，它可能会变成一种有效的药物。然而，由于其复杂的结构，enacloxin很难从头开始合成。与此同时，聚酮化合物合成生物学领域在过去30年中已经发展到生物合成地生产和修饰enacloxin是一种可行的替代方案的程度。合成生物学致力于通过开发和修饰自然界中可用的生物合成机制来构建新的分子。特别地，聚酮酶（PKS）是大自然的非常大的模块化酶组装线，用于具有药用特性的广泛的天然产物，范围从抗肿瘤剂通过降胆固醇剂到抗生素。聚酮衍生分子占最畅销药物的20%，每年全球总收入超过100亿英镑。由于其模块化的性质，PKS可以被有效地修饰以合成新的化合物。基于混合和匹配来自不同装配线的组分的方法非常成功，迄今为止合成了几百个新分子。然而，为了利用这些系统合理生产新化合物，如enacloxin类似物，我们需要了解负责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，我们将获得有关enacloxin生物合成的见解。为了实现这一目标，我们建议研究enacloxin PKS的分子细节，特别是原子分辨率结构，运动和相互作用的组件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的见解，我们建议采用高度互补的解决方案和固态魔角旋转NMR光谱的组合。所提出的方法将使我们能够第一次了解蛋白质的结构如何在从皮秒到毫秒的整个相关范围内在时间尺度上演变。它还将使我们能够直接获得链释放酶和底物携带蛋白质的大型复合物的结构和动力学信息。对这类系统的固态NMR研究将是同类研究中的第一个。该项目将导致对enacloxin生物合成的更好理解，并将使所研究的分子机制能够作为合成生物学和其他化合物合成的通用工具进行部署。这种方法与其他结构生物学方法，如X射线晶体学和cryo-EM，具有很强的互补性。

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.