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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Firefly luciferase catalyzes the bioluminescent oxidation of luciferin in the presence of ATP. The broad field of its application arouses scientific interest in various aspects of the luciferase reaction, including the color of emitted light. Firefly luciferase belongs to the ANL-superfamily and is composed of two domains. The substrates bind to the N-domain (residues 1-445); many residues that are crucial for maintaining the bioluminescence color locate in various parts of its structure. Meanwhile, the C-domain (residues 446-548) remains mostly uninvestigated. We identified the strong red-shifting E457K mutation in the C-domain. The E457 residue is distant from the active site, but highly conserved; it forms a hydrogen bond between an α-helix and a β-sheet of the C-domain. On the basis of the L. mingrelica firefly luciferase with the stabilized N-domain we obtained the following mutants: E457V, E457Q, E457D, E457K, A534R and E457V/A534R. The A534R was designed to mimic a hydrogen bond that E457 and R534 residues form between two α-helixes of the C-domain in certain ANL enzymes. We characterized the initial and mutant enzymes and studied their bioluminescent properties at various temperatures (10-42°C). All the Cdomain mutations were red-shifting, though the effect was pronounced to a variable degree. The bioluminescence spectra were represented as sums of two monomodal spectra, corresponding to putative green and red emitters; the contribution of green emitter increased with the temperature decrease. The simultaneous occurrence of two different emitters implies the coexistence of two forms of luciferase molecule that stabilize them. Considering that the green emitter requires rigid and nonpolar microenvironment, the increase of the red emitter contribution in the mutants demonstrates that the C-domain mutations cause loosening of the active site. Thus, the intact structure of the C-domain is essential for the green bioluminescence of luciferase.