Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Role of E270 in pH- and metal-sensitivities of firefly luciferases.

Firefly luciferases display a typical change in bioluminescence color to red at acidic pH, high temperatures and in the presence of heavy metals. Recently, the proton and metal sensing site responsible for the pH-sensitivity of firefly luciferases, which involves the salt bridges between E311-R337 and H310-E354, was identified. However, it is unclear what other residues contribute to the distinct degrees of pH-sensitivity observed in other firefly luciferases. A multialignment of primary structures of a large set of pH-sensitive and pH-insensitive beetle luciferases showed that the conserved E270 among adult firefly luciferases is substituted by Gly (railroad worms)/Gln (click-beetles) in pH-insensitive ones. Site-directed mutagenesis studies using Macrolampis sp2 and Amydetes vivianii firefly luciferases indeed showed that E270 is important for the pH-dependent activity and spectral profiles: the substitution E270A/G drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. Modelling studies showed that the residue E270 is located in a three-glutamate motif (269EEE271) at the N-terminal of α-helix-10. The results suggest that at acidic pH, the protonation of E270 carboxylate may extend a turn of the helix at the N-terminal, misaligning the pH-sensor and luciferin phenolate binding site residues: S286, I288 and E311. In contrast, the substitution of E270A/G may unwind a turn of the α-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation.