Saturating mutagenesis on the Ser118 residue responsible for the spectral tuning mechanism of the orange/green‐reversible cyanobacteriochrome AM1_1499g1
A. Yoneda, T. Suzuki, R. NarikawaAbstract
Cyanobacteriochromes (CBCRs) are widely distributed photoreceptors in cyanobacteria, crucial for regulating light‐responsive phenomena. CBCR GAF domains form a stable covalent bond with a linear tetrapyrrole chromophore
via
the canonical Cys residue and exhibit reversible photoconversion. The DXCF (Asp‐Xxx‐Cys‐Phe) ‐type CBCRs, a dominant subgroup across various clades, are characterized by a highly conserved second Cys residue involved in both phycocyanobilin (PCB)‐to‐phycoviolobilin (PVB) isomerization and reversible ligation to the chromophore. The AM1_1499g1 GAF domain, categorized as a DXCF‐type CBCR, is unique due to the evolutionary loss of this second Cys residue, which is substituted by Ser118. This domain exhibits an orange/green‐reversible photocycle alongside thermochromism in its photoproduct state, suggesting a novel spectral tuning mechanism to be elucidated.
To elucidate the molecular basis of the spectral tuning mechanism in AM1_1499g1, we performed saturation mutagenesis at the Ser118 position and comprehensively performed spectral analyses of the resulting S118X variants. Spectral analyses classified the S118X mutants into five distinct functional groups based on PCB‐to‐PVB isomerization activity, thermochromism and photocycle property. These results lead us to propose that the side chain property at Ser118, located near the chromophore centre, affects the chromophore conformation, especially the A‐ring conformation, thereby controlling the domain's spectral properties. Framed within the evolutionary transition of AM1_1499g1, our findings demonstrate that single‐site mutagenesis at Ser118 is sufficient to recapitulate a broad range of spectral properties. This variation functionally mimics the natural evolutionary divergence of XRG (extended red/green)‐type CBCR domains, highlighting the exceptional molecular plasticity of this photoreceptor family.