Studies On Cyanobacteriochromes And The Biosynthesis Of Phycoerythrin

Cyanobacteria are prokaryotes with the capability of oxygenic photosynthesis. Under the appropriate aquatic environment, a large amount of algae can grow which can induce water bloom. The process of photosynthesis is initiated by the absorption of light energy through phycobiliproteins. Phycobiliproteins are multi-subunit complexes that covalently bind a variety of linear tetrapyrrole pigments called bilins, enabling them to harvest light in the visible region of the spectrum. Lyases CpcE and CpcF specific for attaching PCB to Cys-84 of CpcA have been identified in several cyanobacteria. PecE and PecF catalyze both the attachment of PCB to PecA and its isomerization. More recently, the lyase CpcS has been identified as one of the lyases attaching PCB to theβ-subunits of CPC and possibly allophycocyanin (ApcA, ApcB, ApcA2, ApcD, ApcF). The lyase CpeTl can catalyze PCB's covalent attachment of theβsubunit Cys-155 of CpcB and PecB.In this study, the entire pathway of the biosynthesis of holo-αandβ-subunits of CPE from Gloeobacter violaceus PCC 7421 was reconstituted in Escherichia coli(E. coli). In PCC 7421, we found 20 genes which are homologous to the active lyase.The genes for the biosynthesis of apo-proteins (apo-CpeA or apo-CpeB), the genes hol and pebA and pebB encoding the enzymes for PEB production and the probable lyase genes for the attachment of PEB to apo-proteins were co-expressed in E. coli by a dual vector system. With the lyase CpeY, holo-asubunits of CPE were acquired which showed spectroscopic properties similar to those of the same protein produced in cyanobacteria. The lyase CpeS2 can partially catalyze the attachment of PEB to CpeB.Zhao and his colleagues found that the lyase from Anabaena sp. PCC7120 can catalyze PCB's covalent attachment ofαsubunit Cys-82 and theβsubunit Cys-80 of CPE from Calothrix sp PCC 7601. CPE in Gloeobacter violaceus PCC 7421 and Calothrix sp PCC 7601 showed high homology. We attempt to find the lyase or the lyase group which can catalyze PCB's covalent attachment of the a subunit Cys-139, theβsubunit Cys-165 or theβsubunit Cys-48/59 of CPE from Calothrix sp PCC 7601.Phytochromes are an important class of light receptors. Cyanobacteriochrome is a special type of phytochromes and phytochrome-related proteins which have been identified in cyanobacteria. Based mainly on sequence homologies and the photochemical properties of some of the proteins reconstituted with bilin chromophores, it is accepted that cyanobacteriochrome is more diverse regarding function, chromophore type and chromophore protein attachment, than the eukaryotic phytochromes of plants and algae. The recombinant molecule overexpressed in E. coli offers greatly improved possibilities for studying phytochrome molecular structure.In this paper, the aphC gene of Anabaena sp. PCC 7120 and coded protein AphC and its GAF domain were studied in detail concerning reconstitution method and function dynamic.Gene aphA and its four site-directed mutagenesis fragments designed by homology analysis were isolated from genomic DNA by PCR using specific primers and DNA polymerase. These site-directed mutagenesis fragments include aphC(C138L), aphA(C478A), aphA(C554L) and aphA(C138L/C554L). The gene fragments were subcloned into pET30a. The pET30a-derived expression vectors were co-expressed with heme oxygenase HO1 and biliverdin reductase PcyA in E. coli. According to the characteristic peaks of the spectra,PCB-AphC shows two distinct reversible conversions。AphC has two distinct chromophore-binding GAF domains, GAF1 domain and GAF3 domain. Isolation of GAF1 domain or GAF3 domain and PCB-producing E. coli demonstrated that it shows reversible photoconversion with a spectrum very similar to the one of PCB-AphC. PCB-GAF1 has been experimentally proved to show reversible photoconversion between red absorbing form (λmax=636nm, Pr) and far-infrared absorbing form (λmax=684nm, Pfr) upon irradiation with 700nm light and 570nm light and PCB-GAF3 shows reversible photoconversion between red absorbing form (λmax=644nm, Pr) and blue absorbing form (λmax =584nm, Pb) upon irradiation with 700nm light and>610nm light. Denatured by acidic urea, the peaks of UV absorption were all at around 662nm. It is indicated that the pigment attached to AphC, GAF1 and GAF3 was PCB. The purified PCB-AphC, PCB-GAF1 and PCB-GAF3 were detected via SDS-PAGE. After stained by Zn2+, they showed fluorescence under UV light, which is characteristic for bilins covalently bound to proteins. TheεandΦF can be obtained by using the data of the fluorescence and absorption spectra.The P(o) state of GAF3 changes in darkness to Pr state rapidly whereas the P(fr) state of GAF1 changes slowly to Pr state in darkness. The full fluorescence spectrum of the P(o) state of GAF3 can be measured at-77K. The CD spectrum of the purified biliproteins PCB-AphC, PCB-AphC(C138L) and PCB-AphC(C554L) in the UV zone showed a-helix structure. Because AphC has a histidine kinase domain in the C-terminal region, we studied the effects of reversible photoconversion on AphC autophosphorylation activity. We observed that the Pr form was less efficiently phosphorylated than the Po form and the Pfr form.