Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
Biochim Biophys Acta Bioenerg. 2020 Oct 1;1861(10):148247. doi: 10.1016/j.bbabio.2020.148247. Epub 2020 Jun 18.
The alternative oxidase (AOX) is a monotopic di‑iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs. In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in V and catalytic efficiency whilst some also affected the K for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SIGNIFICANCE: The alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.
交替氧化酶 (AOX) 是一种单铁羧酸盐蛋白，作为多种植物、真菌和原生动物的末端呼吸链氧化酶。特别重要的是发现，由人类和植物真菌病原体引起的两种新兴传染病，其中大多数对多种药物具有抗药性，似乎都依赖于 AOX 活性才能存活。由于 AOX 不存在于哺乳动物细胞中，因此 AOX 被认为是设计特异性杀真菌和抗寄生虫药物的可行治疗靶标。在这项工作中，我们已经突变了疏水通道内的保守残基（R96、D100、R118、L122、L212、E215 和 T219），晶体学表明这些残基导致了活性部位。我们的数据表明，所有突变都导致 V 和催化效率急剧降低，而有些突变还影响了对苯二酚和氧气的 K 值。还研究了突变对抑制剂敏感性的影响程度，突变 R118 和 T219 导致抑制剂效力完全丧失。然而，当突变 R96 时，仅观察到 IC 值略有降低，这表明该残基在抑制剂结合中不太重要。使用计算机模拟提供了对这种变化原因的深入了解，我们认为这是由于质子转移网络的中断，导致整体反应动力学降低。我们根据泛醌结合位点的结构特征讨论了我们的结果，并考虑了这些发现对催化循环性质的影响。意义：交替氧化酶是一种泛醌氧化还原酶，可催化泛醌的氧化和氧气的还原为水。它广泛分布于植物、真菌和寄生王国，通过促进三羧酸循环的周转，同时减少 ROS 产生，在代谢中发挥核心作用。
