Flavin mononucleotide reductase of luminous bacteria.
NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.
D001728:Dicumarol / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D005486:Flavin Mononucleotide; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010776:Photobacterium / Q000201:enzymology*; D012402:Rotenone / Q000494:pharmacology; D013696:Temperature
D004140:Dinitrophenols; D012402:Rotenone; D005486:Flavin Mononucleotide; D001728:Dicumarol; D008156:Luciferases; D009247:NADH, NADPH Oxidoreductases