Botany, Department I of Biology, Ludwig-Maximilians-University Munich, Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany.
Mol Plant. 2014 Jan;7(1):156-69. doi: 10.1093/mp/sst144. Epub 2013 Nov 6.
Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.
柠檬酸合酶在所有生物体的线粒体三羧酸 (TCA) 循环中起着关键作用，因为它催化第一个关键步骤，即草酰乙酸和乙酰辅酶 A 之间碳-碳键的融合。TCA 循环功能的调节在植物中尤为重要，因为必须协调线粒体活动与光合作用。到目前为止，植物中 TCA 循环活性的翻译后调节几乎完全没有被探索过。尽管已经在体外鉴定出几种 TCA 循环酶是硫氧还蛋白的靶标，但任何已知的卡尔文循环的硫氧还蛋白依赖性调节仍然有待证明。在这里，我们通过对其六个半胱氨酸残基进行定点突变，研究了拟南芥柠檬酸合酶的氧化还原调节。我们的结果表明，氧化通过形成混合二硫键来抑制酶活性，因为部分氧化的柠檬酸合酶形成大的氧化还原依赖性聚集体。此外，我们能够证明硫氧还蛋白可以切割不同的分子内和分子间二硫键，这强烈增强了酶的活性。用酶的半胱氨酸变体进行的活性测量揭示了影响总酶活性以及酶的氧化还原敏感性的重要半胱氨酸残基。
