Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena Sp. PCC7120.
The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-beta84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-beta84 of the apo-beta-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 degrees C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3 microm, and a kcat = 1.7 x 10(-5) and 1.1 x 10(-5) s(-1) for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS.
Zhao
Kai-Hong
KH
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, PR China.
Su
Ping
P
Li
Jian
J
Tu
Jun-Ming
JM
Zhou
Ming
M
Bubenzer
Claudia
C
Scheer
Hugo
H
D017033:Anabaena / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006639:Histidine / Q000737:chemistry; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D052980:Phycobilins; D010798:Phycocyanin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D045725:Tetrapyrroles / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D052980:Phycobilins; D021122:Protein Subunits; D045725:Tetrapyrroles; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin; D006639:Histidine; D008190:Lyases; D003545:Cysteine