Biosynthesis of phycocyanobilin in photosynthetic eukaryotic algae

开链四氢吡咯色素(Bilins)广泛存在于许多生物体中，具有不同的功能。蓝藻、红藻和隐藻是(藻类)胆蛋白浓度最高的生物。在这些生物中，胆红素既用于捕光(藻胆蛋白)，也用于感光(光敏色素)。在这项拨款提案中，我们将研究绿松石藻胆蛋白藻蓝胆素(PCB)的不寻常生物合成途径。虽然蓝藻使用铁氧还蛋白依赖的胆红素还原酶(FDBR)的单一酶在四电子还原步骤中将胆绿素IXα转化为多氯联苯，但一些藻类缺乏这种酶，尽管它们的藻胆蛋白或光敏光敏色素中含有大量的这种色素。例如，属于氰化物的红藻Galdieria Suluraria只在其捕光藻胆蛋白中使用多氯联苯，但缺乏用于多氯联苯的典型生物合成酶。然而，它拥有编码两个FDBR的基因，参与了多氯联苯异构体藻红胆素(PEB)的生物合成。初步数据表明，在这种生物中，PEB确实是多氯联苯的生物合成前体，而且一种迄今未知的异构酶催化了这一转化。因此，这项建议的一个主要目标是通过经典的蛋白质层析和添加生物信息学方法确定推测的候选蛋白质来从硫磺革兰氏菌中纯化这种酶活性。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，我们将调查链柄藻克莱伯索米姆(Klebsormidium FlacciumKlebsormidium Flaccium)植物FDBR HY2的同源物。当高等植物酶以两电子还原的方式将胆绿素IXα转化为植物色动素时，藻类蛋白质正在合成多氯联苯。基于我们最近求解的晶体结构FDBrPebb，生成了KflHY2的结构模型，表明HY2家族中存在天冬氨酸/天冬氨酸开关。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专一性改变的原因，并将确定导致这种令人惊讶的功能变化的其他分子决定因素。由于酶活性中心的三维排列对于解释这些功能数据是必不可少的，我们将集中精力解析KflHY2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.