Lyase activities of CpcS- and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin beta-subunits.
Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins beta-phycoerythrocyanin (PecB) or beta-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine beta155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:alpha-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.
Zhao
Kai-Hong
KH
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China. khzhao@163.com
Zhang
Juan
J
Tu
Jun-Ming
JM
Böhm
Stephan
S
Plöscher
Matthias
M
Eichacker
Lutz
L
Bubenzer
Claudia
C
Scheer
Hugo
H
Wang
Xing
X
Zhou
Ming
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D052980:Phycobilins / Q000096:biosynthesis* / Q000235:genetics; D010798:Phycocyanin / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D052980:Phycobilins; D021122:Protein Subunits; D011994:Recombinant Proteins; C038397:phycoerythrocyanin; D010798:Phycocyanin; D008190:Lyases