Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

Julien Valton,Carole Mathevon,Marc Fontecave,Vincent Nivière,David P Ballou

doi:10.1074/jbc.m709730200

Abstract

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

Highlights

We have been investigating the mechanism of the FMN-dependent twocomponent enzyme system, ActVA-ActVB, which participates in the last steps of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor [11,12,13] (Scheme 1)
The first spectrum recorded at 5 s ([O2] ϭ 595 ␮M after mixing) had a main absorption band at 386 nm. This spectrum is characteristic of a C(4a)-FMNOOH species, such as those found in other single- and two-component flavin-dependent hydroxylases, including HPAH-C2 from A. baumannii [22] and p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa [25, 26]
We have carried out a detailed kinetic study of the reaction of the two-component FMN-dependent monooxygenase, ActVA-ActVB from S. coelicolor, by stopped-flow spectrophotometmonooxygenase by carrying out experiments similar to those ric methods to test our earlier propositions on the reaction described in the previous section with various concentrations of NADϩ

Summary

Introduction

We have investigated the kinetics of the formation and decay of the C(4a)-FMN-OOH intermediate by stopped-flow spectrophotometry at 4 °C, as described under “Experimental Procedures.” An anaerobic solution of FMNred (20 ␮M), containing an excess of ActVA (50 ␮M) to promote full complex formation (Kd FMNred ϭ 0.39 ␮M, [17]), was mixed with equal volumes of Tris-HCl buffer solution containing various concentrations of O2.

Results

Conclusion