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Journal of Biochemistry Advance Access published online on August 30, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm155
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© 2007 The Japanese Biochemical Society

Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues

Chutintorn Suadeea, Sarayut Nijvipakula, Jisnuson Svastia, Barrie Entschb,c, David P. Balloub and Pimchai Chaiyena,*

aDepartment of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; bDepartment of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-06060, USA.; cSchool of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW, 2351, Australia

*Corresponding author: Pimchai Chaiyen, Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400; Tel: 662 201 5607; Fax: 662 354 7174; E-mail: scpcy{at}mahidol.ac.th

Received June 8, 2007; Accepted August 11, 2007


  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°C and pH 8. The measured Kd at 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 t1/2 of 1,020 min while Lux_Vh has t1/2 of 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.

Key Words: bioluminescence, flavin, luciferase, monooxygenase, Vibrio campbellii


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[Abstract] [Full Text] [PDF]


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