FIREFLY LUCIFERASE LUCIOLA MINGRELICA. HISTORICAL ASPECT

The review presents the history of research on the luciferin-luciferase system of fi refl ies Luciola mingrelica at the Department of Chemical Enzymology of MGU, which began in the mid-70s of the last century on the initiative of the fi rst 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 non-stationary enzymatic process, the turnover of the enzyme is very small due to the slow dissociation of the enzyme-product complex. Analysis of the bioluminescence and fl uorescence spectra of the reaction product – oxyluciferin – and its analogues led to the conclusion. that ketoenol tautomers of the phenolate forms of oxyluciferin (ketone, enol and enolate ion) are the most likely emitters in the luciferin-luciferase system of fi refl ies. Native luciferase preparations have been shown to contain phospholipids, the removal of which leads to a decrease in the activity and stability of the enzyme. At the beginning of the 90s, luciferase L. mingrelica 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 fi refl y (67% homology). Using methods of random and site-specifi c 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, 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 our country. Methods of genetic engineering, computer modeling and site-specifi c mutagenesis 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 active site. The superposition of two or three emitter forms fi xed in the bioluminescence spectra indicates the coexistence of various conformational forms of luciferase in the reaction medium, which are in dynamic equilibrium.