Distinctive Properties of Dark Reversion Kinetics between Two Red/Green‐Type Cyanobacteriochromes and their Application in the Photoregulation of cAMP Synthesis

Cyanobacteriochromes (CBCRs) are photoreceptors that bind to a linear tetrapyrrole within a conserved cGMP‐phosphodiesterase/adenylate cyclase/FhlA (GAF) domain and exhibit reversible photoconversion. Red/green‐type CBCR GAF domains that photoconvert between red‐ (Pr) and green‐absorbing (Pg) forms occur widely in various cyanobacteria. A putative phototaxis regulator, AnPixJ, contains multiple red/green‐type CBCR GAF domains. We previously reported that AnPixJ's second domain (AnPixJg2) but not its fourth domain (AnPixJg4) shows red/green reversible photoconversion. Herein, we found that AnPixJg4 showed Pr‐to‐Pg photoconversion and rapid Pg‐to‐Pr dark reversion, whereas AnPixJg2 showed a barely detectable dark reversion. Site‐directed mutagenesis revealed the involvement of six residues in Pg stability. Replacement at the Leu294/Ile660 positions of AnPixJg2/AnPixJg4 showed the highest influence on dark reversion kinetics. AnPixJg2_DR6, wherein the six residues of AnPixJg2 were entirely replaced with those of AnPixJg4, showed a 300‐fold faster dark reversion than that of the wild type. We constructed chimeric proteins by fusing the GAF domains with adenylate cyclase catalytic regions, such as AnPixJg2‐AC, AnPixJg4‐AC and AnPixJg2_DR6‐AC. We detected successful enzymatic activation under red light for both AnPixJg2‐AC and AnPixJg2_DR6‐AC, and repression under green light for AnPixJg2‐AC and under dark incubation for AnPixJg2_DR6‐AC. These results provide platforms to develop cAMP synthetic optogenetic tools.