Mihara Shoko, Yoshida Keisuke, Higo Akiyoshi, Hisabori Toru
Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Japan.
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.
Plant Cell Physiol. 2017 Jan 1;58(1):86-94. doi: 10.1093/pcp/pcw182.
The redox regulation system is widely accepted as a crucial mechanism for controlling the activities of various metabolic enzymes. In addition to thioredoxin reductase/thioredoxin cascades, NADPH-thioredoxin reductase C (NTRC), a hybrid protein formed by an NADPH-thioredoxin reductase domain and a thioredoxin (Trx) domain, is present in chloroplasts and in most cyanobacteria species. Although several target proteins and physiological functions of NTRC in chloroplasts have been characterized, little is known about NTRC functions in cyanobacteria. Therefore, we investigated the molecular basis and physiological significance of NTRC-dependent redox regulation in the filamentous heterocyst-forming nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120). Initially, we identified six candidate NTRC targets in Anabaena 7120 using NTRC affinity chromatography. Subsequently, we compared the efficiency of reducing-equivalent transfer from NTRC and Trx-m1 to the NTRC target protein 2-Cys peroxiredoxin. Biochemical analyses revealed that compared with Trx-m1, NTRC more efficiently transfers reducing equivalents to 2-Cys peroxiredoxin. Subsequently, we constructed and analyzed an ntrC knockout strain in Anabaena 7120. The mutant showed impaired growth under oxidative stress conditions and lower concentrations of reduced 2-Cys peroxiredoxin in cells. Taken together, the present in vitro and in vivo results indicate that NTRC is a significant electron donor for 2-Cys peroxiredoxin and plays a pivotal role in antioxidant defense systems in Anabaena 7120 cells.
氧化还原调节系统被广泛认为是控制各种代谢酶活性的关键机制。除了硫氧还蛋白还原酶/硫氧还蛋白级联反应外，NADPH-硫氧还蛋白还原酶C（NTRC）是一种由NADPH-硫氧还蛋白还原酶结构域和硫氧还蛋白（Trx）结构域组成的杂合蛋白，存在于叶绿体和大多数蓝藻物种中。尽管已经对叶绿体中NTRC的几种靶蛋白和生理功能进行了表征，但对蓝藻中NTRC的功能却知之甚少。因此，我们研究了丝状异形胞形成固氮蓝藻鱼腥藻PCC 7120（鱼腥藻7120）中NTRC依赖性氧化还原调节的分子基础和生理意义。最初，我们使用NTRC亲和色谱法在鱼腥藻7120中鉴定了六个候选NTRC靶标。随后，我们比较了从NTRC和Trx-m1向NTRC靶蛋白2-半胱氨酸过氧化物酶转移还原当量的效率。生化分析表明，与Trx-m1相比，NTRC能更有效地将还原当量转移至2-半胱氨酸过氧化物酶。随后，我们构建并分析了鱼腥藻7120中的ntrC基因敲除菌株。该突变体在氧化应激条件下生长受损，细胞中还原型2-半胱氨酸过氧化物酶的浓度较低。综上所述，目前的体外和体内结果表明，NTRC是2-半胱氨酸过氧化物酶的重要电子供体，在鱼腥藻7120细胞的抗氧化防御系统中起关键作用。
