* To whom correspondence should be addressed.
Received February 7, 2011; Revision received April 4, 2011
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 KmATP and KmLH2 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 KmATP and KmLH2 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.
KEY WORDS: firefly luciferase, Luciola mingrelica, site-directed mutagenesis, cysteine residues, polyhistidine tag, kinetic parameters, thermal stability