A Two-component Flavin-dependent Monooxygenase Involved in Actinorhodin Biosynthesis in Streptomyces coelicolor

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (43), p.44362-44369
• Main Authors: Valton, Julien, Filisetti, Laurent, Fontecave, Marc, Nivière, Vincent
• Format: Article
• Language: English
• Subjects: Anthraquinones - chemistry; Anthraquinones - metabolism; Biochemistry; Biochemistry, Molecular Biology; Dose-Response Relationship, Drug; Escherichia coli - metabolism; Flavins - chemistry; FMN Reductase - chemistry; FMN Reductase - metabolism; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Light; Mixed Function Oxygenases - chemistry; Models, Chemical; Models, Statistical; Open Reading Frames; Oxygen - chemistry; Oxygen - metabolism; Plasmids - metabolism; Spectrophotometry; Streptomyces - enzymology; Streptomyces - metabolism; Streptomyces coelicolor; Thermodynamics; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M407722200

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 (FMN ox and FMN red ) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMN red 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 FMN ox binds to ActVB, where it is reduced, and the resulting FMN red moves to ActVA-ORF5, where it reacts with O 2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.