title: Functional studies of the gene slr2049 from Synechocystis sp. PCC6803 and its site-directed mutation.
abstract: Phycobiliprotein is a homologous family of light-harvesting chromoproteins existing in cyanobacteria, red algae and cryptophytes. Phycobiliprotein is made up of phycobilin and its corresponding apophycobiliprotein, and they are covalently linked by the thioether bond with the bilin lyase. Using the software BLAST, we have found gene slr2049 in Synechocystis sp. PCC6803 homologous to the biliprotein lyase gene cpeS. This paper investigates the protein expressed by gene slr2049 to find the enzymatic activity characteristics. We cloned slr2049 and its related genes cpcB, ho1, and pcyA which are linked with the synthesis of phycocyanin. Special amino acid mutagenesis was performed on slr2049 to construct eight mutants slr2049 (H21S), slr2049 (L23S), slr2049 (A24S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), slr2049 (R107S) and slr2049 (Y124S). These mutants were ligated with vectors pEDFDuet-1 and pET-23a to construct pCDF-cpcB-slr2049 wild-type, pCDF-cpcB-slr2049 mutants and pET-ho1-pcyA, for the purpose of protein expression and analysis. The results showed that the wild-type and mutants slr2049 (H21S), slr2049 (L23S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), and slr2049 (Y124S) can catalyze CpcB to couple on PCB correctly and the products have unique spectral characteristics. However mutants slr2049 (A24S) and slr2049 (R107S) have no spectral characteristics. Thus, it is suggested that alanine at position 24 and arginine at position 107 are the active sites.
authors: College of Life Science, South-central University for Nationalities, Wuhan 430074, China.
authors: Bingjun
authors: B
authors: Liu
authors: College of Life Science, South-central University for Nationalities, Wuhan 430074, China. Electronic address: sili.chen@163.com.
authors: Sili
authors: S
authors: Chen
authors: College of Life Science, South-central University for Nationalities, Wuhan 430074, China.
authors: Lei
authors: L
authors: Zhang
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D052980:Phycobilins / Q000235:genetics / Q000378:metabolism; D010798:Phycocyanin / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D052980:Phycobilins; D010798:Phycocyanin; D001120:Arginine; D000409:Alanine
keywords: Bilin lyase; Phycocyanin; Protein expression; Site-directed mutagenesis; Spectral characteristics; Western blot