Open AccessSpectroscopic and functional characterization of Lampyris turkestanicus luciferase: a comparative study
Author(s) -
Mortazavi Mojtaba,
Hosseinkhani Saman,
Khajeh Khosro,
Ranjbar Bijan,
Emamzadeh A. Rahman
Publication year - 2008
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2008.00411.x
Subject(s) - luciferase , luciferin , luciferases , chemistry , biochemistry , bioluminescence , microbiology and biotechnology , thermostability , enzyme , biology , transfection , gene
Functional expression and spectroscopic analysis of luciferases from Lampyris turkestanicus and Photinus pyralis were carried out. cDNA encoding L. turkestanicus luciferase was isolated by reverse transcription‐polymerase chain reaction, cloned, and functionally expressed in Escherichia coli . The luciferases were purified to homogeneity using Ni‐nitrilotriacetic acid Sepharose, and kinetic properties of luciferase from L. turkestanicus were compared with that from P. pyralis . Amino acid differences in its primary structures in relation to P. pyralis luciferase brought about changes in the kinetic properties of the enzyme as evidenced by substantial lowering of K m for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis‐Menten kinetics with a K m of 95.5 μM for ATP and 20 μM for luciferin. Maximum activity was recorded at pH 8.5, so it might be a suitable reporter for microbial screening at alkaline pH. Tryptophan fluorescence for P. pyralis luciferase was higher than L. turkestanicus luciferase. Substitution of some residues in L. turkestanicus luciferase appears to change the kinetic properties by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by intrinsic and extrinsic fluorescence, Fourier transform infrared spectroscopy, and near‐ultraviolet circular dichroism spectra.