Spectroscopic and functional characterization of Lampyris turkestanicus luciferase: a comparative study.
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 Km for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis-Menten kinetics with a Km of 95.5 muM for ATP and 20 muM 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.
Mortazavi
Mojtaba
M
Department of Biochemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran P. O. Box 14115-175, Iran. saman_h@modares.ac.ir
Hosseinkhani
Saman
S
Khajeh
Khosro
K
Ranjbar
Bijan
B
Emamzadeh
A Rahman
AR
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology*; D008156:Luciferases / Q000737:chemistry* / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D008156:Luciferases