Biosynthesis of phycocyanobilin in photosynthetic eukaryotic algae

开链四吡咯色素（胆素）广泛分布于许多生物体中，具有不同的功能。蓝藻、红藻和隐藻是具有最高浓度（藻）十亿素的生物。在这些生物体中，胆磷脂既用于光收集（藻胆蛋白），也用于光感应（光敏色素）。在这项拨款提案中，我们将研究绿松石藻胆素（PCB）的不同寻常的生物合成途径。虽然蓝藻使用铁氧化还蛋白依赖的胆汁素还原酶（FDBR）中的一种酶在四电子还原步骤中将胆绿素IXalpha转化为PCB，但一些藻类缺乏这种酶，尽管它们的藻胆蛋白或光敏色素中含有大量的这种色素。例如，蓝藻属的一种红藻硫藻（Galdieria suluraria）仅在其捕光藻胆蛋白中使用多氯联苯，但缺乏一种典型的多氯联苯生物合成酶。然而，它具有编码两个FDBRs的基因，这些FDBRs参与pcb异构体藻红蛋白（PEB）的生物合成。初步数据表明，在这种生物体中，PEB确实是PCB的生物合成前体，并且迄今未知的异构酶催化了这种转化。因此，本提案的一个主要目标是通过经典的蛋白质色谱法和生物信息学方法从硫磺草中纯化这种酶活性，并确定可能的候选蛋白质。一旦确定，重组候选蛋白将被纯化，然后进行结构和催化分析。在第二个目标中，我们将研究从链藻Klebsormidium flacacium （KflaHY2）中提取的植物FDBR HY2的同源物。当高等植物酶通过双电子还原将胆绿素IXalpha转化为植物色素移动素时，藻类蛋白正在合成PCB。基于我们最近解决的FDBR PEBB晶体结构，KflaHY2的结构模型被生成，表明在HY2家族中发生了天冬氨酸/天冬氨酸切换。在这里，我们希望通过分析这个外群的几个成员来确认这种氨基酸开关是催化特异性改变的原因，并将确定这种令人惊讶的功能变化的其他分子决定因素。由于酶活性位点的三维排列对这些功能数据的解释至关重要，我们将集中精力解决KflaHY2的晶体结构。

Open-chain tetrapyrrole pigments (bilins) are widely distributed in many organisms where they serve distinct functions. Cyanobacteria, red algae and cryptophytes are organisms with the highest concentration of (phyco)bilins. In these organisms, bilins are employed for both, light-harvesting (phycobiliproteins) and light-sensing (phytochromes). Within this grant proposal we will investigate unusual biosynthetic pathways to the turquoise phycobilin phycocyanobilin (PCB). While cyanobacteria use a single enzyme of the ferredoxin-dependent bilin reductases (FDBR) to convert biliverdin IXalpha in a four-electron reducing step to PCB, some algae lack this enzyme although they contain significant amounts of this pigments in either their phycobiliproteins or in the light sensing phytochromes. The red alga Galdieria sulphuraria, member of the cyanidiales, for instance only uses PCB in its light harvesting phycobiliproteins but lacks a canonical biosynthetic enzyme for PCB. However, it possesses genes encoding two FDBRs involved in the biosynthesis of the PCB-isomer phycoerythrobilin (PEB). Preliminary data suggest that PEB is indeed a biosynthetic precursor to PCB in this organism and that a thus far unknown isomerase catalyses the conversion. One major objective of this proposal is therefore to purify this enzymatic activity from G. sulphuraria by means of classical protein chromatography and with the addition of a bioinformatics approach nail down putative candidate proteins. Once identified, recombinant candidate proteins will be purified followed by structural and catalytic analysis. In a second objective, we will investigate a homolog of the plant FDBR HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). While the higher plant enzyme converts biliverdin IXalpha in a two-electron reduction to phytochromobilin, the algal protein is synthesizing PCB. Based on our recently solved crystal structure the FDBR PEBB, a structural model for KflaHY2 was generated suggesting that an aspartate/aspargine switch occurred within the HY2 family. Here, we wish to confirm that this amino acid switch is the reason for changed catalytic specificity by analyzing several members of this outgroup and will identify other molecular determinants for this surprising change of function. As the three-dimensional arrangement of the enzymes active site is essential for the interpretation of these functional data, we will focus our efforts towards resolving a crystal structure of KflaHY2.