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Journal of Biochemistry Advance Access published online on September 8, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj190
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© 2006 The Japanese Biochemical Society.
Received June 19, 2006
Accepted July 11, 2006

Regular Paper

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

V. R. Viviani 1 *, F. G. C. Arnoldi 2, B. Venkatesh 3, A. J. S. Neto 2, F. G. T. Ogawa 2, A. T. L. Oehlmeyer 2, and Y. Ohmiya 3

1 Universidade Federal de São Carlos, Campus de Sorocaba, Sorocaba, SP, Brazil; Departamento de Biologia Celular e Molecular, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Av. 24 A, 1515, Bela Vista, Rio Claro, SP, Brazil 13506-900
2 Departamento de Biologia Celular e Molecular, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Av. 24 A, 1515, Bela Vista, Rio Claro, SP, Brazil 13506-900
3 Cell Dynamics Research Group, National Institute of Advanced Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka, Japan

* To whom correspondence should be addressed.
V. R. Viviani, E-mail: viviani{at}rc.unesp.br


  Abstract

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. 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.

Keywords: Luciferases; Railroad worms; Phrixotrix; Bioluminescence.
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