Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis.
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 (PACalpha and PACbeta). 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 PACalpha 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 degrees C for the recombinant PACalpha F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACalpha F2 and the F2 domain of the PACalpha homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.
Ito
Shinji
S
Department of Photoscience, School of Advanced Sciences, Graduate University for Advanced Studies (SOKENDAI), Shonan Village, Hayama, Kanagawa 240-0193, Japan.
Murakami
Akio
A
Sato
Kyosuke
K
Nishina
Yasuzo
Y
Shiga
Kiyoshi
K
Takahashi
Tetsuo
T
Higashi
Shoichi
S
Iseki
Mineo
M
Watanabe
Masakatsu
M
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D005056:Euglena gracilis / Q000201:enzymology*; D005415:Flavins / Q000378:metabolism; D005786:Gene Expression Regulation; D008027:Light; D017956:Photoreceptor Cells, Invertebrate / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
D005415:Flavins; D021122:Protein Subunits; D000262:Adenylyl Cyclases