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Journal of Biochemistry Advance Access originally published online on August 30, 2007
Journal of Biochemistry 2007 142(4):539-552; 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{dagger}

Chutintorn Suadee1, Sarayut Nijvipakul1, Jisnuson Svasti1, Barrie Entsch2,3, David P. Ballou2 and Pimchai Chaiyen1,*

1Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; 2Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-06060, USA; and 3School of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW, 2351, Australia

*To whom correspondence should be addressed. 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 1020 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

Abbreviations: C1, reductase component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii; FMNH–, reduced form of FMN; HPA, p-hydroxyphenylacetate; Kd, dissociation constant; kobs, apparent rate constant; Lux, luciferase; Lux_Vc, luciferase from V. campbellii; Lux_Vh, luciferase from V. harveyi; Lux:FMN, complex of luciferase and oxidized FMN; Lux:FMNH–, complex of luciferase and reduced FMN

{dagger} Nucleotide sequence data reported are available in the GenBank database under the accession number EF394780 [GenBank] .


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