Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.
Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.
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DOI：
10.1016/j.bbabio.2016.03.001
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发表时间：
2016-06
期刊：
影响因子：
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通讯作者：
Keisuke Yoshida;T. Hisabori
中科院分区：
文献类型：
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作者：
Keisuke Yoshida;T. Hisabori
线粒体代谢对于维持细胞的生长和维持是重要的;然而，植物线粒体中个体过程的调控机制在很大程度上仍然没有被表征。线粒体苹果酸脱氢酶（mMDH）是三羧酸（TCA）循环和氧化还原穿梭过程中的关键酶，在巯基氧化还原调控下，是硫氧还蛋白（Trx）的候选靶点。此外，腺嘌呤核苷酸状态可能是控制线粒体代谢的另一个因素，因为线粒体中的呼吸ATP产生被认为受到几种环境刺激的影响。利用生物化学和反向遗传的方法，我们解决了氧化还原和腺嘌呤核苷酸依赖的mMDH在拟南芥的调节。重组mMDH蛋白在氧化条件下形成分子内二硫键，但这些键对mMDH活性没有相当大的影响。线粒体定位型Trx（Trx-o）不促进氧化mMDH的再还原。体内氧化还原状态的测定表明，即使在Trx-o-缺陷的植物中，mMDH也以还原形式稳定存在。因此，我们得出结论，mMDH是不是在类氧化还原调节酶。相比之下，腺嘌呤核苷酸（AMP，ADP和ATP）降低mMDH活性。每种腺嘌呤核苷酸以不同的效力抑制mMDH活性，ATP产生最大的抑制作用，Ki显著降低。相应地，mMDH活性被抑制的ATP/ADP比率在生理范围内的增加。这些结果表明，mMDH活性精细控制在线粒体腺嘌呤核苷酸平衡的变化。
Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH inArabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localizedo-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of thein vivoredox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lowerKi. Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.