Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHB314.
To study the assembly of phycocyanin β subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin β subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin β subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the β subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for β subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314.
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China.
Ran
R
Zhang
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China.
Xiao-Ting
XT
Feng
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China.
Fei
F
Wu
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China.
Yan
Y
Ding
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China. Electronic address: xnzang@ouc.edu.cn.
Xiao-Nan
XN
Zang
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003 Shandong, China.
Xue-Cheng
XC
Zhang
National Hybrid Rice Engineering Technology Research Center, Changsha 410125 Hunan, China.
Ding-Yang
DY
Yuan
National Hybrid Rice Engineering Technology Research Center, Changsha 410125 Hunan, China.
Bing-Ran
BR
Zhao
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001654:Bile Pigments / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D008190:Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D054300:Oscillatoria / Q000201:enzymology / Q000235:genetics*; D052980:Phycobilins / Q000378:metabolism*; D010798:Phycocyanin / Q000096:biosynthesis / Q000378:metabolism*; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D001654:Bile Pigments; D004269:DNA, Bacterial; D052980:Phycobilins; D010798:Phycocyanin; C011069:phycocyanobilin; D008190:Lyases
Arthrospira platensis FACHB314; Fluorescence intensity; Site-directed mutation; cpcT