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Natalya N. Ugarova, Galina Yu. Lomakina
Firefly luciferase Luciola Mingrelica. Historical
aspect
Abstract
Abstract. The
review presents the history of research on the luciferin-luciferase system of
fireflies 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
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 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 fluorescence spectra of the reaction product –
oxyluciferin – and its analogues 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, 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 firefly
(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, 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-specific 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 fixed in the bioluminescence
spectra indicates the coexistence of various conformational forms of luciferase
in the reaction medium, which are in dynamic equilibrium.
Key words: bioluminescence, firefly luciferase, Luciola
mingrelica, luciferin, ATP, oxyluciferin, emitter, kinetics, thermal
stability, mutagenesis
Copyright (C) Chemistry Dept., Moscow State University, 2002
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