Biochemistry (Moscow)

, 76:1147 | Cite as

Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase

  • Yu. A. Modestova
  • G. Yu. Lomakina
  • N. N. UgarovaEmail author


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 \(K_m^{LH_2 } \) 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 \(K_m^{LH_2 } \) 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 





inactivation rate constant


fluorescence excitation wavelength


firefly luciferin


polymerase chain reaction


relative light units


initial recombinant luciferase


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Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • Yu. A. Modestova
    • 1
  • G. Yu. Lomakina
    • 1
  • N. N. Ugarova
    • 1
    Email author
  1. 1.Faculty of ChemistryLomonosov Moscow State UniversityMoscowRussia

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