A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays

Abstract

Firefly luciferases have been widely used for bioanalytical purposes during the last 5 decades. They usually emit yellow-green bioluminescence and are pH-sensitive, displaying a color change to red at acidic pH and higher temperature and in the presence of heavy metals. Besides the usual applications as bioanalytical reagents and as reporter genes, firefly luciferases' pH- and metal-sensitivities have been recently harnessed for intracellular metal and pH biosensing. Previously we cloned the luciferase of the Brazilian Amydetes vivianii firefly which displays the most blue-shifted color among known firefly luciferases. Here we purified it, characterized and investigated the kinetic properties and the pH, metal and thermal sensitivities of this firefly luciferase. This luciferase displays the lowest reported K_M for ATP, the highest catalytic efficiencies, and the highest thermostability among the studied recombinant beetle luciferases, making this enzyme and its cDNA an ideal reagent for sensitive ATP assays and reporter gene. The blue-shifted spectrum, higher thermostability, lower pH- and thermal-sensitivities and protein fluorescence studies indicate a more rigid active site during light emission. This enzyme displays an unmatched selective spectral sensitivity for cadmium and mercury, making it a promising ratiometric indicator of such toxic metals. Finally, the weaker thermal-sensitivity compared to other firefly luciferases makes this enzyme a better ratiometric pH indicator at temperatures above 30 °C, suitable for mammalian cell assays.