Understanding of luciferin-luciferase system by the artificial evolution of luciferase

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Site-directed mutagenesis of AbLL, a fatty acyl-CoA synthetase in non-luminous click beetle Agrypnus binodulus, showed that only one-three amino acid substitutions are sufficient to generate the luciferase activity. The activity was weak but the further amino acid changes gave stronger activity, more than 40 times. We also isolated an isotype luciferase, LcLuc2, from the Japanese firefly Luciola cruciata (Oba et al., 2010), suggesting that fireflies possess at least two luciferase genes and that luciferin-binding sites (7 amino acids) in luciferases are highly conserved. Interestingly, the luminescent color by LcLuc2 is pH insensitive, unlike other firefly luciferases.