Chromophore composition and assembly of phycoerythrin III of Prochlorococcus marinus CCMP1375

海洋原绿球藻（Prochlorococcus marinus）是海洋中数量最多、生态意义最重要的光合自养生物之一。与共生的聚藻球菌不同，P. marinus除了具有二乙烯基叶绿素天线外，还具有以称为藻红蛋白III （PE III）的单一藻胆蛋白形式存在的捕光藻胆体的残余物。在适应弱光的海洋p.a marinus CCMP1375生态型中，该PE III由α -亚基和β -亚基与连接的开链四吡啶发色团（藻胆素）组成。光谱学研究推测，该peiii与藻胆素、藻红蛋白（PEB）和藻红蛋白（PUB）以1:3的比例发生色磷酸化。然而，到目前为止，这还没有得到生物化学上的证实，主要是由于生物化学研究的生长产量低。P. marinus CCMP1375中PE组装所需的大部分基因编码在一个约10 kb的基因簇中。该簇不仅包含α -亚基和β -亚基的基因，还包含编码藻胆蛋白裂解酶的基因。这些蛋白质很可能参与了藻胆素与亚基的翻译后附着。此外，在基因簇之外，还可以发现编码藻胆素PEB和藻蓝胆素生物合成酶的基因，以及另外一种假定的藻胆蛋白裂解酶。在这项拨款申请中，我们希望探索peiii的染色质磷酸化状态和组装机制。首先，将通过PE III富集，然后结合紫外可见光谱和荧光光谱以及质谱分析来验证天然PE III的显色反应。使用最近描述的TREX系统，整个基因簇将通过随机转座整合到大肠杆菌BL21 （DE3）基因组后，从侧翼T7启动子双向表达。此外，藻胆素生物合成基因也会在胞外共表达。成功整合和表达后，PE III将被纯化并进行紫外可见和荧光光谱分析，然后对结合的藻胆素进行详细的表征。在并行方法中，我们将使用采用pDuet™矢量系统的模块化表达式。在这里，所有可能参与α或β亚基组装的基因都将共同表达。由于一些假定的藻胆蛋白裂解酶尚未有特征的同源物，因此必须测试不同的组合。此外，第二种方法可以将装配过程分解为各个子单元。该项目的总体目标是确定peiii的色素磷酸化状态，并进一步鉴定和表征所涉及的藻胆蛋白裂解酶。因此，我们也打算鉴定新的藻胆蛋白裂解酶活性，这将有助于理解光捕获结构的组装和这些结构对特定光环境的适应。

The cyanobacterium Prochlorococcus marinus is one of the most abundant and ecologically important photosynthetic autotrophs in the ocean. In contrast to the co-occuring Synechococcus species, P. marinus only possesses remnants of a light-harvesting phycobilisome in form of a single phycobiliprotein termed phycoerythrin III (PE III) in addition to a divinyl-chlorophyll antenna. In the low –light adapted ecotype P. marinus CCMP1375, this PE III consist of an alpha- and beta-subunit with linked open-chain tetrapyrrole chromophores (phycobilins). Spectroscopic studies have postulated that this PE III is chromophorylated with the phycobilins phycoerythrobilin (PEB) and phycourobilin (PUB) at a 1:3 ratio. However, thus far this has not yet been confirmed biochemically mainly due to low growth yields for biochemical studies. Most of the genes required for PE assembly are encoded in a ~ 10 kb gene cluster in P. marinus CCMP1375. This cluster not only contains the genes for the alpha- and beta- subunit respectively, but also genes encoding phycobiliprotein lyases. These are proteins likely to be involved in the posttranslational attachment of phycobilins to the subunits. Furthermore, outside the gene cluster, genes encoding the biosynthetic enzymes for the phycobilins PEB and phycocyanobilin and an additional putative phycobiliprotein lyase can be found. Within this grant proposal, we wish to explore the chromophorylation state and mechanism of assembly of PE III. First, chromophorlyation of native PE III will be validated by PE III enrichment followed by a combination of UV-Vis and fluorescence spectroscopy and mass spectrometry. Using the recently described TREX system, the whole gene cluster will be bidirectionally expressed from flanking T7 promoters following its integration into the Escherichia coli BL21 (DE3) genome using randomized transposition. Additionally, the phycobilin biosynthesis genes will be co-expressed episomally. After successful integration and expression, PE III will be purified and subjected to UV-Vis and fluorescence spectroscopy, followed by detailed characterization of the bound phycobilins. In a parallel approach we will make use of modular expression employing the pDuet™ vector system. Here, all putative genes involved in either alpha- or beta-subunit assembly will be co-expressed. Since some of the putative phycobiliprotein lyases have no characterized homologs yet, different combinations have to be tested. Furthermore, this second approach enables the dissection of the assembly process into the individual subunits. The overall goal within this project is to define the chromophorylation state of PE III and furthermore identify and characterize the involved phycobiliprotein lyases. Thereby we also intent to identify novel phycobiliprotein lyase activities, which will contribute to the understanding of light-harvesting structure assembly and adaptation of those to specific light regimes.