Cyclic nucleotide, such as cyclic GMP, is a secondary messenger that regulates a wide range of biological process via the diverse signaling cascades. Photoactivated adenylyl cyclases (PACs), constituted of blue light utilizing flavin (BLUF) and cyclase homology domain (CHD), are used as an optogenetic tool to modulate the cyclic AMP (cAMP) level and to study cAMP-mediated signal transduction mechanisms. Here, we have engineered photoactivated adenylyl cyclases (PACs) from microbes to photoactivated guanylyl cyclases (PGCs) via mutagenesis of the substrate binding-specific residues in cyclase homology domain. We demonstrate purification, photodynamic, and detailed biochemical characterization of the engineered PGCs that can serve as optogenetic tool for manipulation of cGMP level in the cells. Engineered PGCs show typical BLUF photoreceptor properties with different recovery kinetics and varying light-regulated guanylyl cyclase activities.
Keywords: BLUF domain; Cyclase domain; Optogenetics; Photoactivated adenylyl cyclase (PAC); Photoactivated guanylyl cyclase (PGC); cGMP.