Mechanistic insights in light-induced cAMP production by photoactivated adenylyl cyclase alpha (PACalpha).
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.
Looser
Jens
J
Universität Würzburg, Julius-von-Sachs-Institut, D-97082 Würzburg, Germany.
Schröder-Lang
Saskia
S
Hegemann
Peter
P
Nagel
Georg
G
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D000242:Cyclic AMP / Q000096:biosynthesis*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004789:Enzyme Activation; D005056:Euglena gracilis / Q000201:enzymology / Q000528:radiation effects; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D008027:Light; D009154:Mutation; D055668:Photochemical Processes; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005182:Flavin-Adenine Dinucleotide; D000242:Cyclic AMP; D000262:Adenylyl Cyclases