Xu Fei, Copsey Alice C, Young Luke, Barsottini Mario R O, Albury Mary S, Moore Anthony L
Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Brighton, United Kingdom.
Front Plant Sci. 2021 Oct 22;12:744218. doi: 10.3389/fpls.2021.744218. eCollection 2021.
The alternative oxidase (AOX) is widespread in plants, fungi, and some protozoa. While the general structure of the AOX remains consistent, its overall activity, sources of kinetic activation and their sensitivity to inhibitors varies between species. In this study, the recombinant AOX (rTAO) and AOX1A (rAtAOX1A) were expressed in the Δ mutant FN102, and the kinetic parameters of purified AOXs were compared. Results showed that rTAO possessed the highest and for quinol-1, while much lower and were observed in the rAtAOX1A. The catalytic efficiency ( / ) of rTAO was higher than that of rAtAOX1A. The rTAO also displayed a higher oxygen affinity compared to rAtAOX1A. It should be noted that rAtAOX1a was sensitive to α-keto acids while rTAO was not. Nevertheless, only pyruvate and glyoxylate can fully activate Arabidopsis AOX. In addition, rTAO and rAtAOX1A showed different sensitivity to AOX inhibitors, with ascofuranone (AF) being the best inhibitor against rTAO, while colletochlorin B (CB) appeared to be the most effective inhibitor against rAtAOX1A. Octylgallate (OG) and salicylhydroxamic acid (SHAM) are less effective than the other inhibitors against protist and plant AOX. A Caver analysis indicated that the rTAO and rAtAOX1A differ with respect to the mixture of polar residues lining the hydrophobic cavity, which may account for the observed difference in kinetic and inhibitor sensitivities. The data obtained in this study are not only beneficial for our understanding of the variation in the kinetics of AOX within protozoa and plants but also contribute to the guidance for the future development of phytopathogenic fungicides.
交替氧化酶（AOX）广泛存在于植物、真菌和一些原生动物中。虽然AOX的总体结构保持一致，但其总体活性、动力学激活来源及其对抑制剂的敏感性在不同物种之间存在差异。在本研究中，重组AOX（rTAO）和AOX1A（rAtAOX1A）在Δ突变体FN102中表达，并比较了纯化的AOX的动力学参数。结果表明，rTAO对喹啉-1的Km和Vmax最高，而在rAtAOX1A中观察到的Km和Vmax则低得多。rTAO的催化效率（Vmax/Km）高于rAtAOX1A。与rAtAOX1A相比，rTAO还表现出更高的氧亲和力。值得注意的是，rAtAOX1a对α-酮酸敏感，而rTAO则不敏感。然而，只有丙酮酸和乙醛酸能够完全激活拟南芥AOX。此外，rTAO和rAtAOX1A对AOX抑制剂表现出不同的敏感性，其中抗霉素A（AF）是针对rTAO的最佳抑制剂，而集胞藻黄素B（CB）似乎是针对rAtAOX1A的最有效抑制剂。没食子酸辛酯（OG）和水杨羟肟酸（SHAM）对原生生物和植物AOX的抑制效果不如其他抑制剂。洞穴分析表明，rTAO和rAtAOX1A在疏水腔内衬的极性残基混合物方面存在差异，这可能解释了观察到的动力学和抑制剂敏感性差异。本研究获得的数据不仅有助于我们理解原生动物和植物中AOX动力学的变化，也为未来植物致病真菌杀菌剂的开发提供指导。
