title: The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1.
abstract: Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.
authors.affiliation: Freie Universität Berlin, Pflanzenphysiologie, Königin Luise Strasse 12-16, D-14195 Berlin, Germany. lamparte@zedat.fu-berlin.de
authors.forename: Tilman
authors.initials: T
authors.lastname: Lamparter
authors.forename: Montserrat
authors.initials: M
authors.lastname: Carrascal
authors.forename: Norbert
authors.initials: N
authors.lastname: Michael
authors.forename: Enriqueta
authors.initials: E
authors.lastname: Martinez
authors.forename: Gregor
authors.initials: G
authors.lastname: Rottwinkel
authors.forename: Joaquin
authors.initials: J
authors.lastname: Abian
mesh_terms: D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001664:Biliverdine / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010834:Phytochrome / Q000737:chemistry*; D011485:Protein Binding / Q000235:genetics; D012231:Rhizobium / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; D010446:Peptide Fragments; D010834:Phytochrome; D006639:Histidine; D014357:Trypsin; D003545:Cysteine; D001664:Biliverdine