Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis
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Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis (Iseki et al., Nature, 2002, 415, 1047-1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACα and PACβ). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD" (BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACα by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28-0.32, and the half-life was 34-44 s at 25 °C for the recombinant PACα F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tl10078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACα F2 and the F2 domain of the PACα homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.
- Photochemical and Photobiological Sciences = Photochemical and Photobiological Sciences
Photochemical and Photobiological Sciences = Photochemical and Photobiological Sciences 4(9), 762-769, 2005-09
Royal Society of Chemistry