Role of charge distribution in a flexible loop on bioluminescence color of firefly luciferase: a lesson from nature

Abstract

The presence of Arg353 in PhRE luciferase, which corresponds to the deleted residue in green emitter luciferases, is an important distinctive structural feature of it. Insertion and substitution of positively‐charged amino acids in a flexible loop (352TPEGDDKP359) of firefly luciferase brought about with change of bioluminescence color. Insertion of Arg356 in a green‐emitter luciferase (Lampyris turkestanicus), that is corresponding to Arg353 in P. hirtus, changed the emitted light from green to red. In order to further clarification of the effect of this position in the light shift, residues with positive side‐chain (Arg356 and Lys356) were inserted in Photinus pyralis luciferase which changed the light color to red, while insertion of a residue with negative side‐chain (Glu356) had little effect and a neutral residue (Gln356) had not any effect on color. On the other hand, by insertion and substitution of positive‐charged residues, in the same loop of Lampyris turkestanicus luciferase, different mutation (E354R/Arg356, E354k/Arg356, E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg356 produces a luciferase that emit red light with a single peak bioluminescence spectrum.