Flavin mononucleotide reductase of luminous bacteria

Abstract

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 bacteria. 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 °C, based on a purity estimate of 20%, is 1.7 × 104 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 × 10-5m) and NADPH (Km, 4 × 10-4m) 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 × 10-5m) and FAD (Km, 1.4 × 10-4m) 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 reductaseluciferase complex. © 1975 Dr. W. Junk b.v. Publishers.