Dual‐Cys bacteriophytochromes: intermediates in cyanobacterial phytochrome evolution?

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Abstract
Previous studies have identified three families of knotted phytochrome photoreceptors in cyanobacteria. We describe a fourth type: ‘hybrid’ phytochromes with putative bilin‐binding cysteine residues in both their N‐terminal ‘knot’ extensions and cGMP‐phosphodiesterase/adenylate cyclase/FhlA (GAF) domains, which we designate as dual‐cysteine bacteriophytochromes (DCBs). Recombinant expression of DCBs in Escherichia coli yields photoactive phycocyanobilin (PCB) adducts with red/far‐red photocycles similar to those of the GAF‐Cys‐containing cyanobacterial phytochromes (Cph1s). Incorporation of the PCB precursor, biliverdin IXα (BV), gave multiple populations, one of which appears similar to those of cyanobacterial bacteriophytochromes (cBphPs). A crystal structure of FiDCB bound to BV exhibits two thioether linkages between the GAF‐ and ‘PAS‐knot’‐Cys residues and the C31 and C32 atoms of BV. When expressed in Synechocystis sp. PCC 6803, DCBs incorporate PCB rather than BV. DCBs can be converted to photoactive cBphP‐, Cph1‐, and tandem‐cysteine cyanobacterial phytochrome (TCCP) analogs by removal and/or addition of a cysteine residue by site‐directed mutagenesis. This structural plasticity contrasts with our inability to generate functional photosensor analogs by analogous site‐directed mutagenesis of TCCP and Cph1 representatives. Phylogenetic analysis demonstrates that DCBs do not form a monophyletic clade and also suggest that Cph1 and TCCP families independently emerged from different lineages of cBphPs, possibly via DCB intermediates.
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  • Chungnam National University

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