title: Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues.
abstract: 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.
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Chutintorn
authors.initials: C
authors.lastname: Suadee
authors.forename: Sarayut
authors.initials: S
authors.lastname: Nijvipakul
authors.forename: Jisnuson
authors.initials: J
authors.lastname: Svasti
authors.forename: Barrie
authors.initials: B
authors.lastname: Entsch
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: 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*
chemical_list: D005486:Flavin Mononucleotide; D008156:Luciferases