Abstract:The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACa and PACb. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.cAMP is a ubiquitous second messenger across phyla 1 and multiple adenylyl cyclases, and phosphodiesterases are involved in its formation and degradation, respectively. A light-activated adenylyl cyclase that is crucial for photoavoidance has been identified in the unicellular flagellate Euglena gracilis 2 . This adenylyl cyclase is composed of two PACa and two PACb subunits, which exhibit adenylyl cyclase activity that is enhanced by blue light. Each subunit harbors two BLUF-type photoreceptor domains, binding flavin adenine dinucleotide 3,4 , and two catalytic domains that are homologous to

The flagellate Euglena gracilis contains as photoreceptor complex a heterotetrameric light-sensitive adenylyl cyclase (AC), consisting of the flavoproteins PACalpha and PACbeta. Previously, we demonstrated the functional expression of PACalpha and PACbeta in oocytes from Xenopus laevis and of PACalpha in different animal cell types. Both yielded a blue light-induced increase of cellular [cAMP]. Here, we report that the action spectrum of PACalpha is flavoprotein-typical, with maxima at approximately 380 and approximately 470 nm. Mutational analysis of PACalpha yields a model for its structure and function. PACalpha shows a basal AC activity in the dark which is unaffected by mutating the conserved tyrosines in the two flavin-binding domains (F1, F2), Y60 in F1 and Y472 in F2. Y60 in F1 is, however, essential for photoactivation as light-stimulation of cyclase activity is completely lost in the F1 mutant Y60F. This effect does not occur in the respective mutation in F2 (Y472F). Mutating the two cyclase domains (C1, C2) indicated that C1 and C2 form a heterodimeric catalytic center as in mammalian class III cyclases. Interaction of C1 with C2 in the same molecule could be excluded as coexpression of non-functional C1 and C2 mutants restored light-induced cyclase activity. Our results strongly suggest an intermolecular dimerization of C1 and C2 domains on PACalpha for a functional enzyme.