Probing the molecular basis of oxygen reduction by the alternative oxidases

探索替代氧化酶还原氧的分子基础

基本信息

批准号：
BB/L022915/1
负责人：
Anthony Moore
金额：
$ 34.42万
依托单位：
University of Sussex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL022915%2F1
关键词：
Probing molecular basis oxygen reduction

项目摘要

Enzymes are proteins that facilitate the reactions that enable living organisms to acquire energy for growth, reproduction and maintenance. A key challenge in understanding the structure-function relationship of one such group of enzymes, the alternative oxidases (AOX), rests upon the identification of its substrate and inhibitor-binding site and its mechanism of action. A detailed knowledge of the nature of this binding site is important since it will reveal whether or not there is a common architecture that can be applied to substrate and inhibitor-binding sites in general and hence provide an insight into the mechanism of binding. More importantly, this knowledge will assist in the suitable rational design of phytopathogenic and anti-parasitic drugs that are specifically targeted to the alternative oxidase. It is now recognized that the distribution of the alternative oxidase is substantially wider than previously thought. No longer restricted to plants, some fungi and protists, the alternative oxidase is also widespread amongst human parasites such as Trypanosoma brucei (the causative agent of African sleeping sickness), intestinal parasites such as Cryptosporidium parvum (responsible for an airborne intestinal infection cryptosporidiosis) and opportunistic human pathogens such as Candida albicans (causes candidiasis or 'thrush'). With respect to the role of AOX in fungi, the development of resistance to agrochemicals by plant fungal pathogens is an international problem that affects all major crops. Indeed fungicide resistance is an important factor in the successful cultivation of cereals in the UK. It is estimated that the UK market for fungicides in cereals is approximately £200m (worldwide $3bn) with winter wheat being the main crop. Fungicides are used against a number of diseases, the major one of winter wheat being caused by Septoria tritici. The main chemical classes of fungicides used to treat UK cereals include the sterol biosynthesis inhibitors. The most important and successful group of these fungicides that have proved effective in the control of plant pathogens are the strobilurin fungicides which are specifically targeted to the mitochondrial respiratory chain (Qo site) thereby inhibiting fungal respiration. Unfortunately resistance to this fungicide often develops resulting in an inability to control fungal pathogens through continued application. Although the mechanism for conferring resistance to Qo fungicides is still controversial there is growing evidence to suggest that the addition of inhibitors, such as azoxystrobin, to fungal pathogens results in a strong induction of the alternative oxidase (AOX). AOX is a mitochondrial terminal oxidase which by-passes the Qo site and is induced in all plants, fungal pathogens and protists following stress induction. We have previously demonstrated that fungal plant pathogens such as Septoria tritici, a fungus that causes major leaf spot diseases in wheat & the wheat "Take-all" fungus, Gaeumannomyces graminis var. tritici have the capacity to express an alternative oxidase when treated with respiratory inhibitors thereby allowing a strobilurin-resistant respiratory pathway to develop which may account for the varying efficacy of strobilurin fungicides. The objectives of the present study are to gain further detailed structural knowledge of the mechanism of oxygen reduction, the nature of the protein-ligand interaction and kinetics through the use of mutants and in the presence and absence of inhibitors. Such information will also be important for further catalytic tuning of the alternative oxidases for future gene therapy strategies and will place us in a very powerful position to undertake future rational inhibitor design which will act as specific and potent phytopathogenic and anti-parasitic drugs specifically targeted at the AOX.

酶是促进生物体获取生长、繁殖和维持能量的反应的蛋白质，了解此类酶（即替代氧化酶 (AOX)）的结构与功能关系的一个关键挑战在于识别酶。其底物和抑制剂结合位点及其作用机制的详细知识非常重要，因为它将揭示是否存在可应用于一般底物和抑制剂结合位点的共同结构。从而提供对该机制的深入了解更重要的是，这一知识将有助于专门针对替代氧化酶的植物病原药物和抗寄生虫药物的合理设计。现在人们认识到替代氧化酶的分布比以前想象的要广泛得多。替代氧化酶的存在时间较长，但它在人类寄生虫中也广泛存在，如布氏锥虫（非洲昏睡病的病原体）、肠道寄生虫如小隐孢子虫（引起空气传播的肠道感染隐孢子虫病）和机会性人类病原体，例如白色念珠菌（引起念珠菌病或“鹅口疮”）。关于 AOX 在真菌中的作用，植物真菌病原体对农用化学品的抗性的发展是一个重要因素。据估计，影响所有主要作物的国际性问题确实是影响英国谷物种植的一个重要因素。英国谷物杀菌剂市场规模约为 2 亿英镑（全球 30 亿美元），其中冬小麦是主要作物，杀菌剂用于防治多种病害，其中冬小麦的主要病害是由小麦壳针孢引起的。用于处理英国谷物的杀菌剂包括甾醇生物合成抑制剂，这些杀菌剂中最重要、最成功的一类已被证明可有效控制植物病原体的是嗜球果伞素。杀真菌剂专门针对线粒体呼吸链（Qo位点），从而抑制真菌呼吸，不幸的是，这种杀真菌剂经常产生抗药性，导致无法通过持续使用来控制真菌病原体，尽管对Qo杀真菌剂产生抗性的机制仍然存在争议。越来越多的证据表明，向真菌病原体添加抑制剂（例如嘧菌酯）会导致线粒体末端替代氧化酶 (AOX) 的强烈诱导。氧化酶绕过 Qo 位点，在胁迫诱导后在所有植物、真菌病原体和原生生物中被诱导。我们之前已经证明，小麦壳针孢 (Septoria tritici) 等真菌植物病原体是一种导致小麦和小麦主要叶斑病的真菌。通吃”真菌，小麦全能酵母（Gaeumannomyces graminis var. tritici）在用呼吸抑制剂处理时能够表达替代氧化酶，从而允许耐嗜球果伞素的呼吸途径开发这可能解释嗜球果伞素杀菌剂的不同功效。本研究的目的是通过使用突变体和在氧还原机制、蛋白质-配体相互作用的性质和动力学方面获得更详细的结构知识。这些信息对于进一步催化调整未来基因治疗策略的替代氧化酶也很重要，并且将使我们处于非常有利的地位，以进行未来合理的抑制剂设计，该抑制剂将起到特异性和有效的作用。专门针对 AOX 的植物病原药和抗寄生虫药。