Chutintorn SuadeeSarayut NijvipakulJisnuson SvastiBarrie EntschDavid P. BallouPimchai ChaiyenMahidol UniversityUniversity of Michigan, Ann ArborUniversity of New England Australia2018-08-242018-08-242007-10-01Journal of Biochemistry. Vol.142, No.4 (2007), 539-552175626510021924X2-s2.0-38449118003https://repository.li.mahidol.ac.th/handle/20.500.14594/24110A 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°C and pH 8. The measured Kdat 4°C for the binding of FMNH-to Lux_Vc was 1.8 μM whereas to Lux_Vh, it was 11 μM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2of 1020 min while Lux_Vh has t1/2of 201 min at 37°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. © 2007 The Japanese Biochemical Society.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1093/jb/mvm155