Biosynthesis of phycocyanobilin in photosynthetic eukaryotic algae

开链四吡咯色素（胆色素）广泛分布在许多生物体中，它们发挥不同的功能。蓝细菌、红藻和隐藻是具有最高浓度的（藻）胆素的生物。在这些生物体中，胆色素用于捕光（藻胆蛋白）和感光（光敏色素）。在这项拨款提案中，我们将研究不寻常的生物合成途径，蓝绿色藻胆素藻蓝胆素（PCB）。虽然蓝细菌使用铁氧还蛋白依赖性胆色素还原酶（FDBR）的单一酶在四电子还原步骤中将胆绿素IX α转化为PCB，但一些藻类缺乏这种酶，尽管它们在藻胆蛋白或感光光敏色素中含有大量这种色素。例如，蓝藻目（cyanidiales）的成员硫藻（Galdieria sulphuraria）仅在其捕光藻胆蛋白中使用多氯联苯，但缺乏多氯联苯的典型生物合成酶。然而，它具有编码参与PCB异构体藻红胆素（PEB）的生物合成的两个FDBR的基因。初步数据表明，聚乙二醇确实是一种生物合成的前体，在这种生物体中的多氯联苯和迄今未知的异构酶催化的转换。因此，本建议的一个主要目的是从G.通过经典的蛋白质层析，并与生物信息学方法钉下来推定的候选蛋白质。一旦鉴定，将纯化重组候选蛋白，然后进行结构和催化分析。在第二个目标中，我们将研究来自链霉菌Klebsormidium flaccidum（KflaHY 2）的植物FDBR HY 2的同源物。当高等植物酶将胆绿素IX α在双电子还原中转化为植物色素移动素时，藻类蛋白质正在合成PCB。基于我们最近解决的FDBR PEBB晶体结构，产生了KflaHY 2的结构模型，表明在HY 2家族内发生了天冬氨酸/精氨酸转换。在这里，我们希望通过分析该外群的几个成员来确认这种氨基酸开关是改变催化特异性的原因，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于解释这些功能数据至关重要，我们将集中精力解决KflaHY 2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.