The Research On Function Of Gene Slr2049 From Synechocystis Sp. PCC 6803

Phycobiliproteins are a homologous family of light-harvesting chromoproteins existed in cyanobacteria, red algae and cryptophytes, and of which phycocyanin is the one. It follows that phycobiliproteins play a very important role in the process of the photosynthesis. At present, water eutrophication is one of the severest pollutions in our country, and many kinds of algae should be responsible for that problem. Moreover, phycobiliproteins have commercial value according to some recent studies. Therefore, to do in-depth research on the biosynthesis pathway of phycobiliproteins is not only good for the environmental protection and enhancement, but also is a big economic benifit. Phycobiliprotein is made up of phycobilin and its corresponding apophycobiliprotein, and they are covalently linked by the thioether bond. In vivo, the correct attachment of most chromophores is catalyzed by lyases. The lyase genes cpcE and cpcF which were found in the Synechococcus sp. PCC7002 are the first studied lyase genes, and we find that cpcE and cpcF have got high homology in many other cyanobacterias. In recent years, there are many reports proving that. For example, in 2006, Zhao, et al. have found a biliprotein lyase gene cpeS (alr0617) in the in the cyanobacterium, Anabaena (Nostoc) PCC7120. The protein which is encoded by gene cpeS can catalyze the phycocyanobilin to attach to cysteine-84 of theβ-subunit of phycobiliprotein(PC) or phycoerythrocyanin(PEC) .In this paper, we have found gene slr2049 in Synechocystis sp. PCC 6803 which is homologous to the biliprotein lyase gene cpeS by some softwares for homologous analysis. We cloned three other genes from the genome of Synechocystis sp. PCC 6803: heme oxygenase gene1 (ho1), ferredoxin oxidoreductase gene (pcyA) andβ-phycocyanin gene (cpcB). These three genes were cloned to investigate the function of the protein encoded by gene slr2049, in order to know if the protein Slr2049 had the same or similar function to biliprotein lyase CpeS. We constructed the expressive plasmids: pCDF-cpcB-slr2049 and pET-ho1-pcyA, then, in the heterologous E. coli system, recombined the phycocyanobilin andβ-phycocyanin which were detected by absorption spectra and SDS-PAGE.The results showed that the protein encoded by gene slr2049 had the function of biliprotein lyase.On the other hand, in order to research the function of the protein encoded by gene slr2049 deeply, we constructed two site-directed mutants: slr2049 (W14L) and slr2049 (Y132S). We also did the same procedures as the wild type with the two mutants. The results showed that Slr2049 (W14L) still had the catalytic ability, but the ability lowered so much than the wild type, while Slr2049 (Y132S) had almost the same catalytic ability as the wild type. So we can infer that the tryptophan at the fourteenth site has active effects to the catalytic ability of the protein, but the tyrosine at the 132 site hasn't.