Luciola mingrelica Firefly Luciferase: Historical Aspect

Abstract

This review presents the history of research on the luciferin–luciferase system of fireflies Luciola mingrelica at the Division of Chemical Enzymology, Department of Chemistry, Moscow State University, which began in the mid-1970s at the initiative of the first head of the Department, Professor I.V. Berezin. Based on the study of the kinetics of enzymatic oxidation of luciferin, a kinetic scheme of the reaction was proposed, according to which in an aqueous solution the luciferase reaction is a nonstationary enzymatic process and the turnover of the enzyme is very small due to the slow dissociation of the enzyme–product complex. Analysis of the bioluminescence and fluorescence spectra of the reaction product oxyluciferin and its analogs led to the conclusion that keto–enol tautomers of the phenolate forms of oxyluciferin (ketone, enol and enolate ion) are the most likely emitters in the luciferin–luciferase system of fireflies. Native luciferase preparations have been shown to contain phospholipids, whose removal leads to a decrease in the activity and stability of the enzyme. At the beginning of the 1990s, L. mingrelica luciferase was cloned. The enzyme in the primary sequence turned out to be close to other luciferases of the genus Luciola, cloned in Japan (more than 80% homology), but differed from the previously studied luciferase from American P. pyralis fireflies (67% homology). Using methods of random and site-specific mutagenesis, a library of mutant forms of L. mingrelica luciferase with altered bioluminescence spectra (green and red luciferases) was created. Thermostable mutants of luciferase were obtained by the method of directed evolution, and in particular, a highly active and thermostable mutant (4TS), on the basis of which an ATP-reagent was developed, which is still widely used in bioluminescent analysis by many researchers in Russia. Genetic engineering, computer modeling and site-specific mutagenesis methods have been used to clarify the role of the dynamic structure of the enzyme in the complex, three-stage oxidation of the luciferin. It has been shown that the emitter (electronically excited oxyluciferin) is an intramolecular label in the enzyme’s active site. The superposition of two or three emitter forms fixed in the bioluminescence spectra indicates the coexistence of various conformational forms of luciferase in the reaction medium, which are in dynamic equilibrium.