Research On Lyases Catalyzing The Attachment Of Phycocyanobilin To Phycobiliprotein

As we know now, CpcE/F is the lyase catalyzing the attachment of phycocyanobilin to phycobiliprotein, but the mechanism is still unknown. In this work, we aimed to validate this proposal and to map in more detail the functions to the protein sequence, using the ways of deletion mutation, site-directed mutations . What's more, Since there was no report about the lyase for Cys-81 ofα-APC and Cys-82 ofβ-APC, our work mainly deals with the finding and characterization of this (or these) lyase. Some gene fragments was found by homology analyses, these gene fragments were cloned and expressed in E.coli for further research.Through analyzing CpcE/F and PecE/F, seven deletion mutants: CpcF(1-160), CpcF(10-213), CpcF(I9K), CpcE(1-274), CpcE(L276D), CpcE(1-272), CpcE(L275D) of the CpcE/F of the cyanobacterium, Mastigocladus laminosus PCC7603, were constructed to probe the functional domains. A 53 aa truncation in CpcF C-terminus caused a loss of the ability to form a complex with CpcE, CpcF(I9K) had not influence to activity of the lyase, CpcE(1-272) had only 17~22% activity of the wild type,possibly due to misfolding. the deletion interferes with the refolding of CpcE. According to the activity of CpcE(L275D) and CpcE(L276D),L276 is a critical residue.Through the experimences ofα-APC andβ-APC in the reconstitution system in vitro, it can be proved that the reaction was mostly quenched by the detergent Triton X-100 and EDTA. The best reaction conditions were 0.2mM mercaptoethanol,70mM Mg2+,800~1000mM Phosphate buffer(pH8.0). The optimum reaction temperature was 30~37°C. It can't succeed in the reconstitution system ofβ-APC in vitro and only partly succeeded when reconstitution with PCB-lyase-84. Maybe it was because of the construction of PCB.