Biosynthesis, properties and structure of phytochrome photoreceptors from cyanobacteria

Abstract

Recombinant cyanobacterial phytochrome CphA from Tolypothrix sp. PCC 7601 (formerly Calothrix) was shown to bind the linear tetrapyrroles phytochromobilin (PΦB), phycocyanobilin (PCB) and phycoerythrobilin (PEB). PCB- and PΦB adducts formed photochromic species displaying U.V./visible spectral properties characteristic of cyanobacterial and higher plant phytochromes, with the non-photochromic PEB-CphA adduct displaying the expected fluorescence spectrum. Kinetic analyses for the binding of linear tetrapyrroles to CphA has revealed dissociation and catalytic rate constants that are similar to those determined for higher plant phytochromes (for binding of PEB to CphA K_d = 3.22 μM, k_cat = 3.4x10^-4 s^-1). Circular dichroism studies suggest that a change in the secondary structure of holo-CphA may be occurring during photoisomerisation from the red light-absorbing (P_r) form to the far-red absorbing (P_fr) form. Purified PCB-CphA appears to exist as a multimer of approximately 2 MDa. This is contrary to other known phytochromes that exist as dimers of approximately 170 kDa in cyanobacteria or approximately 250kDa in higher plants. Crystallographic trials were performed with recombinant PCB-CphA from Tolypothrix sp. PCC 7601.

Haem oxygenases, HO1 from Synechocystis sp. PCC 6803 and HO1 from Arabidopsis thaliana, were recombinantly expressed for use in structural studies. Synechocystis HO1 was shown to be irreversibly inhibited in the presence of divalent transition metal ions. Crystallographic trials were performed with HO1 from Synechocystis sp. PCC 6803 and with HO1 from Arabidopsis thaliana.

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