Selection of Phrixothrix hirtus luciferase mutants (Coleoptera: Phengodidae) with higher catalytic constants and affinities for luciferin and ATP to the development of more efficient luciferases
Luciferases are enzymes responsible for bioluminescence. These enzymes catalyze the exergonic highly oxidation of substrates known as luciferins, that the energy is released as light with high efficiency. The firefly luciferase are the most known and used in the biotechnology field as ATP bioindicators reagents, biosensors, cell biomarkers, reporter genes, among other applications. In the biomedical field, luciferase genes from fireflies have been used to study the bacterial and viral progression and regression and tumor regression in animal models. The luciferases from North American, European and Japanese fireflies have been the most used in these proposals; however, they have limited applicability because they produce yellow-green bioluminescence, which is partially absorbed by hemoglobin and bone tissues. Phrixothrix hirtus luciferase, cloned and characterized by our group, is the one that naturally emits red light, and has a high affinity for luciferin and ATP, being potentially useful for analytical testing and pigmented samples. However, this enzyme has low catalytic constant and quantum yield when compared to green light luciferases. Based on prior knowledge of the structure and function of this and other luciferases, in this project we aim to develop a red light emitting luciferase with increased catalytic efficiency, reducing the KM for the substrates and increasing the catalytic constant and possibly improve the quantum yield by techniques of site-directed and random mutagenesis. The results will be correlated with the structure of the enzyme. We also plan investigate the applicability of this enzyme as bioanalytical reagent in pigmented samples. (AU)
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