Color-tuning of Firefly Luciferase Bioluminescence by Modification of Enzyme and Substrate Structure: New Opportunities for Optical Imaging

Luciferase from Photinus pyralis (PpyLuc) has been extensively investigated and knowledge about mechanism of light emission of bioluminescent insects is mainly due to the studies related to structure and function of this enzyme. PpyLuc is, at present, the preferred enzyme for applications in optical molecular imaging because of favourable characteristics of the emitted yellow-green light (λmax 560 nm). Red and far-red emissions from a firefly luciferase could greatly improve the detection in small animal imaging, considerably reducing tissue attenuation of emitted light. Several bioluminescent insects, such as click beetle and railroad worm, possess luciferases that use the same substrates as PpyLuc but are able to emit red light. Results of researches aimed to shift light emission of PpyLuc towards the red emission by modification of the structure of the enzyme or that of the substrate have been here reviewed. The three-dimensional structure of PpyLuc has been solved in the absence of substrates or inhibitors, and, therefore, luciferin binding-site residues have been suggested by modeling studies or by means of site-directed mutagenesis. A few residues randomly distributed over the primary structure of firefly luciferases are known to affect bioluminescence colors and their substitution has resulted in red mutants. Chemical modifications of the substrate are limited in number and a few ATP and luciferin analogues have been proven to be able to shift light emission from yellow-green to red.