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

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. , 7), in the utilization of sulfur from aliphatic sulfonates in E. coli (8), and in the desulfurization of fossil fuels by Rhodococcus species (9), as well as the hydroxylation of 4-hydroxyphenylacetate in Acinetobacter baumannii (10). 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 (11-13) (Scheme 1).The first enzyme of the two-component flavin-dependent oxygenases is a NAD(P)H:flavin oxidoreductase that catalyzes the reduction of free oxidized flavins (FAD and/or FMN) by the reduced pyridine nucleotides, NADPH or NADH (3,[14][15][16]. The second enzyme is an oxygenase that binds the resulting free reduced flavin and promotes its reaction with O 2 to hydroxylate the substrate (2,3,(5)(6)(7)(8)(9)(10)17). Therefore, in contrast to the single-component flavin hydroxylases, the two-component sys-* This work was supported by National Institutes of Health Grant GM64711 (to D. P. B.). 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. 1 To whom correspondence may be addressed.

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

“…Examples for the former include 4-hydroxyphenyl acetate 3-monooxygenase (HpaB) of Escherichia coli (21) and 2,4,5-trichlorophenol monooxygenase from Burkholderia cepacia (22). FMN-dependent systems are involved in the synthesis of antibiotics such as actinorhodin in Streptomyces coelicolor (23), the bacterial degradation of polyaminocarboxylates such as nitrilotriacetic acid (24), and the desulfurization of fossil fuels by rhodococci (25). pHPAH (EC 1.14.13.3), a well characterized FMN-utilizing enzyme from Acinetobacter baumanii, catalyzes the hydroxylation of p-hydroxyphenyl acetate (HPA) to 3,4-dihydroxyphenyl acetate (PDB entry 2JBT) (26).…”

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

“…For example, IsdG and IsdI share structural similarity to Streptomyces monooxygenases used in antibiotic synthesis, such as ActVA of Streptomyces coelicolor (23). However, the oxidoreductase ActVB oxidizes NADH to NAD ϩ , whereas it reduces FMN and then transfers reduced FMN to ActVA, which in turn re-oxidizes FMN during catalysis (78). No homolog of ActVB could be identified by a BLASTP analysis of the genome of S. aureus strain Newman.…”

Structure of nitrilotriacetate monooxygenase component B fromMycobacterium thermoresistibile

“…The reaction mechanism of this class of enzymes has been a topic of discussion in recent years (Filisetti et al, 2003;van den Heuvel et al, 2004;Valton et al, 2008). One group proposed the ping-pong mechanism, while others described the reactions as following an ordered sequential mechanism.…”

“…Valton et al (2008) alternative carbon sources in the human host. Specifically, M. tuberculosis is able to persist in the glucose-deficient and fatty-acid-rich environment found in the macrophage stage of infection, thereby contributing to its unparalleled virulence (Savvi et al, 2008).…”

“…1. Sequences were selected either based on structural homology or owing to well reported functional similarity (Valton et al, 2008;Filisetti et al, 2003). MthNTA-MoB shares many conserved residues within this family of short-chain flavin reductases, including Arg12, Gly35, Pro48, Leu76, Phe88, His130 and Leu152 (Fig.…”