Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.
The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.
Viviani
V R
VR
Universidade Federal de Sao Carlos, Campus de Sorocaba, Sorocaba, SP, Brazil. viviani@rc.unesp.br
Arnoldi
F G C
FG
Venkatesh
B
B
Neto
A J S
AJ
Ogawa
F G T
FG
Oehlmeyer
A T L
AT
Ohmiya
Y
Y
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D001517:Coleoptera / Q000201:enzymology*; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D019476:Insect Proteins; D008156:Luciferases