Brazilian Beetle luciferases 10 years later: a journey from biodiversity, through structure and function, to bioimaging and biosensing

摘要

Among known luciferases, beetle luciferases are the only ones that can produce a wide variety of colors, ranging from green to red. Among them, fi refl y luciferases from the Northern hemisphere were extensively studies under the structural and functional points of view, and were extensively used as bioanalytical reagents and reporter genes. However, until the last decade, railroadworms and click beetles luciferases, that produce the widest range of colors and are pH-insensitive were poorly studied. In the past 10 years, our group cloned several new beetle luciferases from Brazilian fauna, and investigated their structure and function relationships, and developed new applications with them: the Pyrearinus termitilluminans larval click beetle luciferase[1],which emit the most blue-shifted bioluminescence color (λmax = 534 nm) among beetle luciferases, the green and red emitting luciferases of Phrixotrix spp railoradworms[2], the latter being the only true natural red emitting luciferase (λmax = 623 nm), and Cratomorphus (λmax = 548 nm)[3] and Macrolampis (λmax = 564/600 nm)[4] fi refl y luciferases. These luciferases were characterized and used as models to investigate the relationship between structure, bioluminescence colors and pH-sensitivity[5]. Kinetic studies showed that fi refl y luciferases and Phrixotrix red emitting luciferase display fl ash-like kinetics and the lowest KM values for luciferin, whereas the green emitting click beetle and railroadworm luciferases display slower kinetics and higher KM values for luciferin. The catalytic constants were higher for fi refl y and for Pyrearinus click beetle luciferaseTs. Among these luciferases, Pyrearinus termitilluminans was the most thermostable, and proved to be suitable for cell in vivo assays.