Abstracts

Firefly luciferases are pH-sensitive: their bioluminescence spectra changes from yellow-green to red in the presence of heavy metals, acidic pH or at higher temperatures. These enzymes are being prospected as color tuning and ratiometric biosensors of intracellular pH and toxic metals. The luciferase of Amydetes vivianii firefly is highly efficient, thermostable and has a spectral selectivity for cadmium and mercury, making it a very promising bioanalytical reagent. The residues involved in the pH-sensing and metal binding are H310, E311, R337 and E354, which form two salt bridges, one closer to the phenolate group of oxyluciferin (E311/R337) and another more external (H310/E354). However, considering that some pH-sensitive luciferases display substitution at position 310, the specific role of H310 in pH and metal sensing is still under debate. Thus, the aim of this work was to understand the specific role of the H310 residue in the pH- and metals sensitivities using Amydetes vivianii firefly luciferase and to obtain better suited mutants for biosensing purposes. Site-directed mutagenesis using Phusion TM High-Fidelity DNA Polymerase (Thermo Fisher) was performed, the luciferase mutants were expressed in E. coli BL-21, the mutant enzymes were purified by affinity chromatography using Ni-NTA-Agarose resin and their kinetics, bioluminescence spectra and pH- and metal sensitivities were characterized. The ab initio modeling was performed using the I-TASSER program. MIB2 modeling server was used to screen the bivalent ion site interactions with the luciferase models. Nine mutants of H310 (C, D, E, F, G, Q, T, R and Y) were obtained. There was no significant change of the spectrum of all these mutants at pH 8.0. However, the negatively charged mutants (H310D e H310E) increased the spectral shift at pH 6.0, as well as the sensitivity to metals. Only H310F led to a significant decrease in sensitivity to pH and metals. The results indicate that the presence of negatively charged and basic side-chains at position 310 are important for pH-sensitivity and metals coordination, but not essential, indicating that the remaining side-chains of E311 and E354 may still play a role in pH-sensitivity and coordinate some metals. Furthermore, the effect of metals on bioluminescence activity and modelling studies highlighted the existence of additional metal binding sites with high affinity for Zn2+, Ni2+ and Hg2+, which are unaffected by the substitutions of H310.