title: The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin.
abstract: The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.
authors.affiliation: Department of Life Sciences (Biology), The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan.
authors.forename: Takami
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kamiya
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Rei
authors.initials: R
authors.lastname: Narikawa
authors.forename: Takumi
authors.initials: T
authors.lastname: Noguchi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kohchi
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Inomata
authors.forename: Masahiko
authors.initials: M
authors.lastname: Ikeuchi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D000458:Cyanobacteria / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D052980:Phycobilins / Q000737:chemistry* / Q000378:metabolism; D010798:Phycocyanin / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001426:Bacterial Proteins; D052980:Phycobilins; C526232:phycoviolobilin; D010798:Phycocyanin; C011069:phycocyanobilin