A two-component flavin-dependent monooxygenase involved in actinorhodin biosynthesis in Streptomyces coelicolor.
The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.
Valton
Julien
J
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DRDC-CEA/CNRS/UniversitéJoseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble 9, France.
Filisetti
Laurent
L
Fontecave
Marc
M
Nivière
Vincent
V
D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D038181:FMN Reductase / Q000737:chemistry* / Q000378:metabolism; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008956:Models, Chemical; D015233:Models, Statistical; D016366:Open Reading Frames; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism; D013816:Thermodynamics; D014466:Ultraviolet Rays; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D038181:FMN Reductase; D005415:Flavins; D006899:Mixed Function Oxygenases; D010100:Oxygen; C013390:actinorhodin; D000095622:Benzoisochromanequinones