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Journal of Bacteriology, March 2008, p. 1531-1538, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01660-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence{triangledown}

Sarayut Nijvipakul,1 Janewit Wongratana,1 Chutintorn Suadee,1 Barrie Entsch,2,3 David P. Ballou,2 and Pimchai Chaiyen1*

Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,1 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109,2 School of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW 2351, Australia3

Received 13 October 2007/ Accepted 13 December 2007

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 µM, 2.7 µM, and 1.7 s–1, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH– for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH– for the luminescence reaction in vivo.

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

{triangledown} Published ahead of print on 21 December 2007.

Journal of Bacteriology, March 2008, p. 1531-1538, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01660-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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