Site-Directed Mutagenesis of Cysteine Residues of Luciola mingrelica Firefly Luciferase

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Yu. A. Modestova, G. Yu. Lomakina, and N. N. Ugarova^*

Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-2660; E-mail: nugarova@gmail.com

^* To whom correspondence should be addressed.

Received February 7, 2011; Revision received April 4, 2011

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Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His₆-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K_m^ATP and K_m^LH2 values were increased by a factor of ~1.5-1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K_m^ATP and K_m^LH2 values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ~2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.

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KEY WORDS: firefly luciferase, Luciola mingrelica, site-directed mutagenesis, cysteine residues, polyhistidine tag, kinetic parameters, thermal stability

DOI: 10.1134/S0006297911100087

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