Abstract：
Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.
摘要：
植物色素是植物中存在的胆红素光感受器，藻类,某些细菌和真菌。陆地植物植物色素使用植物色素素（PΦB）作为胆色素发色团。链球菌藻类的植物色素，陆地植物进化的进化枝，使用藻蓝菌素(PCB)，导致更蓝移的吸收光谱。两种发色团均由铁氧还蛋白依赖性胆绿素IXα（BV）开始合成。在蓝细菌和绿藻中，BV被FDBR藻蓝蛋白：铁氧还蛋白氧化还原酶（PcyA）还原为PCB，然而,在陆地植物中，BV被植物色素合酶（HY2）还原为PΦB。然而,系统发育研究表明，链藻藻类中不存在PcyA的任何直系同源物，并且仅存在PΦB生物合成相关基因（HY2）。链霉藻Klebsormidiumnitens（以前的Klebsormidiumflaccidum）的HY2已被间接表明参与PCB生物合成。这里,我们在大肠杆菌中过表达并纯化了K.nitensHY2（KflaHY2）的His6标记变体。采用厌氧胆色素还原酶活性测定和耦合植物色素组装测定，我们确认了产物并鉴定了反应的中间体。定点诱变揭示了两个对催化至关重要的天冬氨酸残基。虽然不可能通过简单地交换催化对将KflaHY2转化为产生PΦB的酶，对HY2谱系另外两个成员的生化研究使我们能够定义两个不同的进化枝，PCB-HY2和PΦB-HY2进化枝。总的来说,我们的研究深入了解了FDBR的HY2谱系的进化。
