![]()
|
|
||||||||
J. Biol. Chem., Vol. 279, Issue 43, 44362-44369, October 22, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||


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 (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.
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. 14.
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.
![]()
CiteULike
Complore
Connotea
Del.icio.us
Digg
Reddit
Technorati What's this?
This article has been cited by other articles:
![]() |
J. Valton, C. Mathevon, M. Fontecave, V. Niviere, and D. P. Ballou Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor J. Biol. Chem., April 18, 2008; 283(16): 10287 - 10296. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Sucharitakul, P. Chaiyen, B. Entsch, and D. P. Ballou Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii J. Biol. Chem., June 23, 2006; 281(25): 17044 - 17053. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Valton, M. Fontecave, T. Douki, S. G. Kendrew, and V. Niviere An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase: THE ActVA-ActVB SYSTEM FROM STREPTOMYCES COELICOLOR J. Biol. Chem., January 6, 2006; 281(1): 27 - 35. [Abstract] [Full Text] [PDF] |
||||
![]() |
P. C. Dorrestein, E. Yeh, S. Garneau-Tsodikova, N. L. Kelleher, and C. T. Walsh Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis PNAS, September 27, 2005; 102(39): 13843 - 13848. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Yeh, S. Garneau, and C. T. Walsh Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis PNAS, March 15, 2005; 102(11): 3960 - 3965. [Abstract] [Full Text] [PDF] |
||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |