Probing the molecular basis of oxygen reduction by the alternative oxidases

酶是促进反应的蛋白质，使生物体能够获得生长、繁殖和维持所需的能量。了解一组这样的酶，替代氧化酶（AOX）的结构-功能关系的一个关键挑战在于其底物和底物结合位点及其作用机制的鉴定。这种结合位点的性质的详细知识是重要的，因为它将揭示是否有一个共同的架构，可以适用于一般的底物和底物结合位点，因此提供了一个洞察结合的机制。更重要的是，这方面的知识将有助于适当的合理设计的植物病原体和抗寄生虫药物，专门针对替代氧化酶。现在认识到，替代氧化酶的分布比以前认为的要宽得多。替代氧化酶不再局限于植物、一些真菌和原生生物，也广泛存在于人类寄生虫如布氏锥虫（非洲昏睡病的病原体）、肠道寄生虫如隐孢子虫（导致空气传播的肠道感染隐孢子虫病）和机会性人类病原体如白色念珠菌（导致念珠菌病或“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影响所有主要作物的国际问题。事实上，杀菌剂抗性是英国成功种植谷物的重要因素。据估计，英国谷物杀菌剂市场约为2亿英镑（全球30亿美元），冬小麦是主要作物。杀真菌剂用于防治许多疾病，冬小麦的主要疾病是由三叶壳针孢引起的。用于处理英国谷物的杀菌剂的主要化学类别包括甾醇生物合成抑制剂。已证明在控制植物病原体中有效的这些杀真菌剂中最重要和最成功的一组是嗜球果伞素杀真菌剂，其特异性靶向线粒体呼吸链（Qo位点），从而抑制真菌呼吸。不幸的是，对这种杀真菌剂的抗性经常发展，导致无法通过持续施用来控制真菌病原体。尽管赋予对Qo杀真菌剂的抗性的机制仍有争议，但越来越多的证据表明，向真菌病原体中添加抑制剂如嘧菌酯导致对交替氧化酶（AOX）的强烈诱导。AOX是一种线粒体末端氧化酶，它绕过Qo位点，在所有植物、真菌病原体和原生生物中在胁迫诱导后被诱导。我们以前已经证明，真菌植物病原体，如壳针孢，一种真菌，导致主要的叶斑病在小麦和小麦“全蚀”真菌，小麦全蚀病菌，禾顶囊壳变种。当用呼吸抑制剂处理时，嗜球果伞素具有表达替代氧化酶的能力，从而允许嗜球果伞素抗性呼吸途径的发展，这可以解释嗜球果伞素杀真菌剂的不同功效。本研究的目的是通过使用突变体以及在存在和不存在抑制剂的情况下获得有关氧还原机制、蛋白质-配体相互作用性质和动力学的进一步详细结构知识。这些信息也将是重要的，为未来的基因治疗策略的替代氧化酶的进一步催化调整，并将使我们在一个非常强大的位置，进行未来的合理的抑制剂设计，这将作为具体的和有效的植物病原体和抗寄生虫药物，特别是针对AOX。

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.