Isoform-Specific NO Synthesis by Arabidopsis thaliana Nitrate Reductase
Isoform-Specific NO Synthesis by Arabidopsis thaliana Nitrate Reductase
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DOI：
10.3390/plants8030067
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发表时间：
2019-03-16
期刊：
影响因子：
4.5
通讯作者：
Fischer-Schrader, Katrin
中科院分区：
文献类型：
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作者：
Mohn, Marie Agatha;Thaqi, Besarta;Fischer-Schrader, Katrin
硝酸盐还原酶（NR）是高等陆地植物的重要酶，它催化硝酸盐同化途径中的限速步骤，即硝酸盐还原为亚硝酸盐。此外，它被认为是重要信号分子一氧化氮（NO）的主要酶源，其在亚硝酸盐的单电子还原中产生。与许多其他植物一样，模式植物拟南芥表达NR的两种亚型（NIA1和NIA2）。到目前为止，只有NIA2是详细的生化研究的焦点，而NIA1等待生化表征。在这项研究中，我们表达和纯化的NIA1的功能片段，并进行各种生化测定与相应的NIA2片段进行比较。我们分析了动力学参数，在多个稳态试验中使用硝酸盐或亚硝酸盐作为底物，并测量底物消耗（硝酸盐或亚硝酸盐）或产物形成（NO）。我们的研究结果表明，NIA1是更有效的亚硝酸盐还原酶，而NIA2表现出更高的硝酸盐还原酶活性，这支持的假设，异构体在植物中有特殊的功能。此外，我们成功地恢复了生理电子传递途径的还原型烟酰胺腺嘌呤二核苷酸（NADH）和硝酸盐或亚硝酸盐为底物，通过混合NR的N-和C-末端片段，从而开辟了新的可能性，研究NR的活性，调节和结构。
Nitrate reductase (NR) is important for higher land plants, as it catalyzes the rate-limiting step in the nitrate assimilation pathway, the two-electron reduction of nitrate to nitrite. Furthermore, it is considered to be a major enzymatic source of the important signaling molecule nitric oxide (NO), that is produced in a one-electron reduction of nitrite. Like many other plants, the model plant Arabidopsis thaliana expresses two isoforms of NR (NIA1 and NIA2). Up to now, only NIA2 has been the focus of detailed biochemical studies, while NIA1 awaits biochemical characterization. In this study, we have expressed and purified functional fragments of NIA1 and subjected them to various biochemical assays for comparison with the corresponding NIA2-fragments. We analyzed the kinetic parameters in multiple steady-state assays using nitrate or nitrite as substrate and measured either substrate consumption (nitrate or nitrite) or product formation (NO). Our results show that NIA1 is the more efficient nitrite reductase while NIA2 exhibits higher nitrate reductase activity, which supports the hypothesis that the isoforms have special functions in the plant. Furthermore, we successfully restored the physiological electron transfer pathway of NR using reduced nicotinamide adenine dinucleotide (NADH) and nitrate or nitrite as substrates by mixing the N-and C-terminal fragments of NR, thus, opening up new possibilities to study NR activity, regulation and structure.