Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis

There is extensive research which explores the photochemistry of Blue Light Using FAD photoreceptors, however there is limited literature available on how the signal is transduced from the BLUF domain to the functional/output domain. A detailed understanding of their operation is needed to direct applications such as the development of optogenetic devices. Here, we elucidate the signal transduction pathway of the photoactivated adenylate cyclase OaPAC, in which a BLUF domain is fused to an adenylate cyclase domain that catalyzes the light-dependent formation of cAMP from ATP. OaPAC has very low dark state activity and is therefore a promising starting point for optogenetic applications. We used unnatural amino acid mutagenesis to explore how light absorption by the N-terminal BLUF domain on the fs timescale leads to activation of the C-terminal adenylate cyclase domain. We incorporated the IR probe azidophenylalanine at F103, W90 and F131 using orthogonal aminoacyl-tRNA-synthetases. The FTIR light minus dark difference spectrum shows that the environment around F103 and W90 residues changes upon light activation, however, there is no change in the environment around F131 upon photoactaction. The enzymatic activity of these mutants was not affected due to the IR probe incorporation. We also replaced Y125 with fluorotyrosine analogues to increase the acidity of the phenolic hydroxyl group and probe the role of Y125 in signal transduction which forms an intersubunit hydrogen bond with N256 from the other monomer in the OaPAC dimer. OaPAC with 3-fluorotyrosine, 3,5-difluorotyrosine or 2,3,5-trifluorotyrosine incorporated at Y125 showed 10 fold higher activity in the dark compared to wild-type OaPAC, whereas Y125F and Y125S showed no dark state activity, implying that the intersubunit hydrogen bond between Y125 and N256 is critical for signal transduction.