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  • The transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs via free diffusion.

The transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs via free diffusion.

Biochemistry (2013-09-06)
Ruchanok Tinikul, Warintra Pitsawong, Jeerus Sucharitakul, Sarayut Nijvipakul, David P Ballou, Pimchai Chaiyen
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.

MATERIALS
Product Number
Brand
Product Description

Riboflavin sodium phosphate, European Pharmacopoeia (EP) Reference Standard
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Riboflavin 5′-monophosphate sodium salt hydrate, synthetic, ≥70% (HPLC)
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Riboflavin 5′-monophosphate sodium salt hydrate, 73-79% (fluorimetric)
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Riboflavin 5′-monophosphate sodium salt hydrate, suitable for electrophoresis, suitable for acrylamide photopolymerization, ≥70%, powder
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Riboflavin Sodium Phosphate Hydrate, tested according to Ph. Eur.