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J. Biol. Chem., Vol. 279, Issue 43, 44362-44369, October 22, 2004

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

Julien Valton, Laurent Filisetti, Marc Fontecave, and Vincent Nivière

From the Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DRDC-CEA/CNRS/UniversitéJoseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France

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 O₂ to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.

Received for publication, July 9, 2004 , and in revised form, August 3, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1–4.

To whom correspondence may be addressed. Tel.: 33-4-38-78-91-03; Fax: 33-4-38-78-91-24; E-mail: mfontecave{at}cea.fr. To whom correspondence may be addressed. Tel.: 33-4-38-78-91-09; Fax: 33-4-38-78-91-24; E-mail: vniviere{at}cea.fr.

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