LuxG is a functioning flavin reductase for bacterial luminescence

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

^* To whom correspondence should be addressed. Email: scpcy{at}mahidol.ac.th.

	Abstract

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 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 as 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, K_m for NADH, K_m for FMN, and k_catwere 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 when compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH^- for the luminescence reaction in vivo.