VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, 40 Thorvaldsensvej, 1871 Frederiksberg C, Denmark.
Plant Physiol Biochem. 2012 Sep;58:89-97. doi: 10.1016/j.plaphy.2012.06.017. Epub 2012 Jun 28.
In chloroplasts, the ferredoxin/thioredoxin pathway regulates enzyme activity in response to light by reduction of regulatory disulfides in target enzymes, ensuring coordination between photosynthesis and diurnal metabolism. Although earlier studies have suggested that many starch metabolic enzymes are similarly regulated, redox regulation has only been verified for a few of these in vitro. Using zymograms and enzyme assays, we performed a comprehensive analysis of the redox sensitivity of known starch metabolising enzymes in extracts of Arabidopsis thaliana. Manipulation of redox potentials revealed that several enzymatic activities where activated by reduction at physiologically relevant potentials. Among these where the isoamylase complex AtISA1/AtISA2, the limit dextrinase AtLDA, starch synthases AtSS1 and AtSS3, and the starch branching enzyme AtBE2. The reversibility of the redox reaction was confirmed by enzyme assays for AtLDA, AtSS1 and AtSS3. Analysis of an AtBAM1 knock-out mutant identified an additional redox sensitive β-amylase activity, which was most likely AtBAM3. A similar requirement for reducing conditions was observed for recombinant chloroplastic α-amylase (AtAMY3) activity. This study adds further candidates to the list of reductively activated starch metabolising enzymes and supports the view that redox regulation plays a role in starch metabolism.
在叶绿体中，铁氧还蛋白/硫氧还蛋白途径通过还原靶酶中的调节二硫键来调节酶活性，以确保光合作用和昼夜代谢之间的协调。尽管早期的研究表明许多淀粉代谢酶也受到类似的调节，但这些酶中的少数几种在体外已经得到了氧化还原调节的验证。本研究使用同工酶谱和酶活性测定法，对拟南芥提取物中已知的淀粉代谢酶的氧化还原敏感性进行了全面分析。氧化还原电势的操纵表明，在生理相关的电势下，几种酶活性被还原激活。其中包括同工淀粉酶复合物 AtISA1/AtISA2、极限糊精酶 AtLDA、淀粉合酶 AtSS1 和 AtSS3 以及淀粉分支酶 AtBE2。通过对 AtLDA、AtSS1 和 AtSS3 的酶活性测定，证实了氧化还原反应的可逆性。对 AtBAM1 敲除突变体的分析鉴定了另一种对氧化还原敏感的β-淀粉酶活性，很可能是 AtBAM3。重组质体α-淀粉酶（AtAMY3）活性也需要还原条件。这项研究增加了可还原激活的淀粉代谢酶的候选物，并支持氧化还原调节在淀粉代谢中起作用的观点。
