title: Phytochrome assembly. The structure and biological activity of 2(R),3(E)-phytochromobilin derived from phycobiliproteins.
abstract: The unicellular rhodophyte, Porphyridium cruentum, and the filamentous cyanobacterium, Calothrix sp. PCC 7601, contain phycobiliproteins that have covalently bound phycobilin chromophores. Overnight incubation of solvent-extracted cells at 40 degrees C with methanol liberates free phycobilins that are derived from the protein-bound bilins by methanolytic cleavage of the thioether linkages between bilin and apoprotein. Two of the free bilins were identified as 3(E)-phycocyanobilin and 3(E)-phycoerythrombilin by comparative spectrophotometry and high pressure liquid chromatography. Methanolysis also yields a third bilin free acid whose absorption and 1H NMR spectra support the assignment of the 3(E)-phytochromobilin structure. This novel bilin is the major pigment isolated from cells that are pre-extracted with acetone-containing solvents. Since phytochrome- or phytochromobilin-containing proteins are not present in either organism, the 3(E)-phytochromobilin must arise by oxidation of phycobilin chromophores. This pigment is not obtained by similar treatment of a cyanobacterium and a rhodophyte that lack phycoerythrin. Therefore, 3(E)-phytochromobilin appears to be derived from phycoerythrobilin-containing proteins. Comparative CD spectroscopy of 3(E)-phytochrombilin and 3(E)-phycocyanobilin suggests that the two bilins share the R stereochemistry at the 2-position in the reduced pyrrole ring. Incubation of 2(R),3(E)-phytochromobilin with recombinant oat apophytochrome yields a covalent bilin adduct that is photoactive and spectrally indistinguishable from native oat phytochrome isolated from etiolated seedlings. These results establish that the phycobiliprotein-derived 2(R),3(E)-phytochromobilin is a biologically active phytochrome chromophore precursor.
authors.affiliation: Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
authors.forename: J
authors.initials: J
authors.lastname: Cornejo
authors.forename: S I
authors.initials: SI
authors.lastname: Beale
authors.forename: M J
authors.initials: MJ
authors.lastname: Terry
authors.forename: J C
authors.initials: JC
authors.lastname: Lagarias
mesh_terms: D001664:Biliverdine / Q000031:analogs & derivatives* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D045342:Light-Harvesting Protein Complexes; D009682:Magnetic Resonance Spectroscopy; D000432:Methanol; D010834:Phytochrome / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011487:Protein Conformation; D000461:Rhodophyta / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D045342:Light-Harvesting Protein Complexes; D010940:Plant Proteins; D010834:Phytochrome; C075811:phytochromobilin; D001664:Biliverdine; D000432:Methanol