title: The transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs via free diffusion.
abstract: Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH(-)) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH(-) to serve as a luciferin in its reaction. However, FMNH(-) is unstable and can react with oxygen to generate H2O2, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH(-) to luciferase in vivo. In this report, the mode of transfer of FMNH(-) between LuxG from Photobacterium leiognathi TH1 and LuxABs from both P. leiognathi TH1 and Vibrio campbellii (PlLuxAB and VcLuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of p-hydroxyphenylacetate hydroxylase (C2) from Acinetobacter baumannii, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH(-) from LuxG. With all FMNH(-) acceptors used (C2, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH(-) with a rate constant of 4.5-6 s(-1). Our data showed that the kinetics of binding of FMNH(-)to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNH(-) or FMNH(-) generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH(-) to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH(-) from LuxG to either LuxAB.
authors.affiliation: Mahidol University , Nakhonsawan Campus, Nakhonsawan 60130, Thailand.
authors.forename: Ruchanok
authors.initials: R
authors.lastname: Tinikul
authors.forename: Warintra
authors.initials: W
authors.lastname: Pitsawong
authors.forename: Jeerus
authors.initials: J
authors.lastname: Sucharitakul
authors.forename: Sarayut
authors.initials: S
authors.lastname: Nijvipakul
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D004058:Diffusion; D005486:Flavin Mononucleotide / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism*; D015394:Molecular Structure; D010088:Oxidoreductases / Q000378:metabolism*; D010776:Photobacterium / Q000201:enzymology; D014733:Vibrio / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C063122:luxG protein, Bacteria; D008156:Luciferases