The Transfer of Reduced Flavin Mononucleotide from LuxG Oxidoreductase to Luciferase Occurs via Free Diffusion

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• 作者：Ruchanok Tinikul ; Warintra Pitsawong ; Jeerus Sucharitakul ; Sarayut Nijvipakul ; David P. Ballou ; Pimchai Chaiyen
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：October 1, 2013
• 年：2013
• 卷：52
• 期：39
• 页码：6834-6843
• 全文大小：486K
• 年卷期：v.52,no.39(October 1, 2013)
• ISSN：1520-4995

文摘

Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNHp>鈥?/sup>) 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 FMNHp>鈥?/sup> to serve as a luciferin in its reaction. However, FMNHp>鈥?/sup> 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 FMNHp>鈥?/sup> to luciferase in vivo. In this report, the mode of transfer of FMNHp>鈥?/sup> 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 FMNHp>鈥?/sup> from LuxG. With all FMNHp>鈥?/sup> acceptors used (C₂, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNHp>鈥?/sup> with a rate constant of 4.5鈥? sp>鈥?p>. Our data showed that the kinetics of binding of FMNHp>鈥?/sup>to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNHp>鈥?/sup> or FMNHp>鈥?/sup> generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNHp>鈥?/sup> 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 FMNHp>鈥?/sup> from LuxG to either LuxAB.