Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues.
A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH(-)). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4 degrees C and pH 8. The measured Kd at 4 degrees C for the binding of FMNH(-) to Lux_Vc was 1.8 microM whereas to Lux_Vh, it was 11 microM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2 of 1020 min while Lux_Vh has t1/2 of 201 min at 37 degrees C. The superior thermostability and tighter binding of FMNH(-) make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before.
Suadee
Chutintorn
C
Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Nijvipakul
Sarayut
S
Svasti
Jisnuson
J
Entsch
Barrie
B
Ballou
David P
DP
Chaiyen
Pimchai
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability / Q000502:physiology; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010776:Photobacterium / Q000201:enzymology; D011485:Protein Binding / Q000502:physiology; D040681:Structural Homology, Protein; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology*
D005486:Flavin Mononucleotide; D008156:Luciferases