Identification Of Phycocyanobilin And Phycobiliprotein From Cyanobacteria

Phycobiliproteins are light-harvesting proteins present in cyanobacteria. The most important step in phycobilin biosynthesis is the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed by binding-site and chromophore-specific lyases, of which only few have hitherto been characterized.By homology analyses, the protein encoded by gene alr0617 was proved to be the lyase of Cys-84 ofβ-CPC andβ-PEC, Cys-82 ofα-APC andβ-APC. Name is CpeS.The gene cpcB from Anabaena PCC 7120 were cloned with vector and verified by sequencing. The overexpressed encoded protein by cpcB were direct used to reconstitution with PCB in vitro. As a result, the better expressive condition is: temperature is 35°C, time is 6 hours and LB culture medium.With CpeS, the holophycobiliproteins PCB-β-CPC and PCB-β-PEC were synthesized in vivo reconstitution. But PCB-β-PEC(C84S) were not obtained. Without CpeS, the productivity of PCB-β-CPC is 11.8% and PCB-β-PEC is 6%.It was shown that CpeS is the lyase of Cys-84 ofβ-CPC andβ-PEC.To study the structure-function relationships and the effect of the histidine residues to the CpeS of phycocyanin, CpeS(H22V) mutants were constructed. The lyase activity of these mutants were tested by reconstitution in vivo to study the effects of histidine to CpeS. Comparing with the wild CpeS, the lyase activity of CpeS(H22V) is 14.5%.The phycocyanobilin peptides were obtained from the naturalβ-CPC,β-PEC, APC and reconstituted PCB-CpcB(C155I),PCB-PecB ( C155I ) ,PCB-α-APC,PCB-β-APC hydrolyzed by pepsin, respectively. After denaturation in the dark with urea in the presence of hydrochloric acid (pH=2), the PCB was not destroyed.With HPLC, we make a comparison of the retention time between the natural and the reconstituted peptides. The results showed that CpeS catalyzes PCB attachment sites at Cys-84 inβ-CPC andβ-PEC, Cys-82 inα-APC andβ-APC. APC were consist ofα-APC andβ-APC.