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Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity

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Abstract

Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0–7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.

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Abbreviations

DLSA:

5′-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine

DTT:

dithiothreitol

E:

luciferase

LO:

oxyluciferin

PCR:

polymerase chain reaction

λmax :

maximum of bioluminescence spectra

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Authors and Affiliations

  1. Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia

    M. I. Koksharov & N. N. Ugarova

Authors
  1. M. I. Koksharov
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  2. N. N. Ugarova
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Correspondence to N. N. Ugarova.

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Original Russian Text © M. I. Koksharov, N. N. Ugarova, 2008, published in Biokhimiya, 2008, Vol. 73, No. 8, pp. 1071–1080.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM07-441, July 6, 2008.

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Koksharov, M.I., Ugarova, N.N. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity. Biochemistry Moscow 73, 862–869 (2008). https://doi.org/10.1134/S0006297908080038

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  • DOI: https://doi.org/10.1134/S0006297908080038

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