Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein.
The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300-14305). We now show that CpeS1 can bind PCB, as assayed by Ni(2+) chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower ( approximately 1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1-PCB complex.
Tu
Jun-Ming
JM
Department Biologie I-Botanik, Universität München, Menzinger Str. 67, 80638, München, Germany.
Kupka
Michaela
M
Böhm
Stephan
S
Plöscher
Matthias
M
Eichacker
Lutz
L
Zhao
Kai-Hong
KH
Scheer
Hugo
H
D001059:Apoproteins / Q000378:metabolism*; D055162:Biocatalysis; D008190:Lyases / Q000378:metabolism*; D052980:Phycobilins / Q000378:metabolism*; D010798:Phycocyanin / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D052980:Phycobilins; D010798:Phycocyanin; C011069:phycocyanobilin; D008190:Lyases