The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive
The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive
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DOI：
10.1093/mp/ssp011
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发表时间：
2009-05-01
期刊：
影响因子：
27.5
通讯作者：
Javier Cejudo, Francisco
中科院分区：
文献类型：
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作者：
Manuel Perez-Ruiz, Juan;Gonzalez, Maricruz;Javier Cejudo, Francisco
NADPH硫氧还蛋白还原酶C(NTRC)是一种能结合NADPH硫氧还蛋白还原酶(NTR)和硫氧还蛋白(TRX)活性的叶绿体酶，可有效还原2-半胱氨酸(2-Cys)过氧化还蛋白(2-Cys Prx)。由于NADPH可以在黑暗中在叶绿体中产生，NTRC在植物夜间过氧化氢解毒中起着关键作用。结果表明，NTRC的四元结构与其氧化还原状态密切相关。在体外，大多数酶以寡聚体的状态存在，在加入NADPH、NADH或DTT后以二聚体的形式分解。凝胶过滤和Western印迹分析表明，天然NTRC形成了对NADPH和DTT敏感的聚集体，这表明聚集状态可能是体内NTRC活性的一个重要方面。此外，该酶定位于拟南芥叶绿体中的簇状结构。取代NADPH结合位点关键残基的NTRC三重和双重突变体A164G-V182E-R183F和A164G-R183F活性降低，但仍能二聚，但中间形式增加。基于这些结果，我们认为NTRC的催化活性形式是二聚体，它的形成是由NADPH诱导的。
NADPH thioredoxin reductase C (NTRC) is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase (NTR) and thioredoxin (TRX) activities for the efficient reduction of 2-Cys peroxiredoxin (2-Cys PRX). Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G-V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.