Mechanism and regulation of the two-component FMN-dependent monooxygenase ActVA-ActVB from Streptomyces coelicolor
Mechanism and regulation of the two-component FMN-dependent monooxygenase ActVA-ActVB from Streptomyces coelicolor
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DOI：
10.1074/jbc.m709730200
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发表时间：
2008-04-18
影响因子：
4.8
通讯作者：
Ballou, David P.
中科院分区：
文献类型：
--
作者：
Valton, Julien;Mathevon, Carole;Ballou, David P.
天蓝色链霉菌ActVA-ActVB系统是一个双组分黄素依赖性单加氧酶，参与抗生素放线菌紫素的生物合成。ActVB是NADH：黄素氧化还原酶，其提供还原的FMN为ActVA，ActVA是催化放线菌紫素前体二氢卡拉芬净羟基化的单加氧酶。本文采用停流分光光度法研究了ActVA催化二氢卡拉芬净羟基化反应的机理以及ActVB还原FMN转移到ActVA的机理。结果表明，羟基化反应机理是在ActVA活性中心有两种不同的反应中间体参与。首先，在还原的FMN与单加氧酶结合并与O-2反应后形成C（4a）-FMN-氢过氧化物物质。该中间体使底物羟基化并转化为第二反应中间体，C（4a）-FMN-羟基物质。此外，我们证明了还原的FMN可以有效地从还原酶转移到单加氧酶，而不涉及任何蛋白质。发现还原的FMN从ActVB转移到ActVA的速率受从ActVB释放NAD（+）的控制，并受NAD（+）浓度的强烈影响，IC 50为40 μ M。NAD（+）对还原FMN转移的这种控制与在ActVB的活性位点中NAD（+）和还原FMN之间形成强电荷转移复合物有关。这些结果表明，在天蓝色链霉菌中，还原酶组分ActVB可以通过控制还原的FMN向单加氧酶的转移来作为单加氧酶活性的调节组分。
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 IC50 of 40 mu M. 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.