Mechanistic insights in light-induced cAMP production by photoactivated adenylyl cyclase alpha (PAC alpha)

作者：Looser Jens; Schroeder Lang Saskia; Hegemann Peter; Nagel Georg^*

来源：Biological Chemistry, 2009, 390(11): 1105-1111.

DOI：10.1515/BC.2009.132

• cystic fibrosis transmembrane conductance regulator (CFTR)
• Euglena gracilis
• FAD
• oocyte
• two-electrode voltage-clamp

The flagellate Euglena gracilis contains as photoreceptor complex a heterotetrameric light-sensitive adenylyl cyclase (AC), consisting of the flavoproteins PAC alpha and PAC beta. Previously, we demonstrated the functional expression of PAC alpha and PAC beta in oocytes from Xenopus laevis and of PAC alpha in different animal cell types. Both yielded a blue light-induced increase of cellular [cAMP]. Here, we report that the action spectrum of PAC alpha is flavoprotein-typical, with maxima at similar to 380 and similar to 470 nm. Mutational analysis of PAC alpha yields a model for its structure and function. PAC alpha 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 PAC alpha for a functional enzyme.

• 出版日期2009-11