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Journal of Biochemistry Advance Access originally published online on September 8, 2006
Journal of Biochemistry 2006 140(4):467-474; doi:10.1093/jb/mvj190
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© 2006 The Japanese Biochemical Society.

ARTICLE

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

V.R. Viviani1,2,*, F.G.C. Arnoldi2, B. Venkatesh3, A.J.S. Neto2, F.G.T. Ogawa2, A.T.L. Oehlmeyer2 and Y. Ohmiya3

1 Universidade Federal de Sao Carlos, Campus de Sorocaba, Sorocaba, SP, Brazil; 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; and 3 Cell Dynamics Research Group, National Institute of Advanced Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka

* To whom correspondence should be addressed at: 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. Tel: +55 19 35264149, Fax: +55 19 35236502, E-mail: viviani{at}rc.unesp.br

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.


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