The cyanide-resistant alternative oxidases from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme.
The cyanide-resistant alternative oxidases from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme.
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来自真菌树干毕赤酵母和粗糙脉孢菌的抗氰化物替代氧化酶是单体，缺乏植物酶的调节特征。
DOI：
10.1006/abbi.2000.1834
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发表时间：
2000
影响因子：
3.9
通讯作者：
J. Siedow
中科院分区：
文献类型：
--
作者：
A. L. Umbach;J. Siedow
植物和真菌的线粒体都有抗氰化物的替代氧化酶，这些氧化酶使用线粒体泛素池中的还原剂将氧还原为水，而这种反应不为ATP合成节省能量。二聚体植物替代氧化酶在其亚基由二硫键连接时相对不活跃。当这种键被削弱时，酶就会被它的激活剂——α -酮酸刺激。蛋白质n端部分的一个Cys负责这两个特征。我们检测了从两种真菌粗神经孢子菌和毕赤酵母分离的线粒体中选择性氧化酶的二聚体结构、形成分子间二硫化物的能力以及对α -酮酸的敏感性。这两种真菌替代氧化酶都不能被二胺共价连接，这在邻近的Cys残基之间诱导形成二硫键，也不能被赖氨酸特异性试剂或戊二醛完全交联的浓度的植物替代氧化酶二聚体交联。α -酮酸丙酮酸和乙醛酸不刺激真菌线粒体的替代氧化酶活性。当琥珀酸盐是呼吸底物时，丙酮酸盐确实刺激活性，但这对替代氧化酶没有直接影响。相比之下，添加GMP是真菌替代氧化酶活性的强激活剂。对植物和真菌选择性氧化酶蛋白序列的分析发现，植物序列中约有40个氨基酸的独特结构域围绕着真菌序列中不存在的调节Cys。这个结构域可能是植物酶二聚化发生的地方。与植物酶相比，本文研究的真菌替代氧化酶是单体的，其活性不依赖于α -酮酸。
Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis. The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond. When this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids. A Cys in the N-terminal section of the protein is responsible for both of these features. We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to alpha-keto acids. Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely. Alternative oxidase activity in fungal mitochondria was not stimulated by the alpha-keto acids pyruvate and glyoxylate. Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase. In contrast, added GMP was a strong activator of fungal alternative oxidase activity. Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences. This domain may be where dimerization of the plant enzymes occurs. In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of alpha-keto acids.