Julien Valton
Aromatic hydroxylation reaction catalyzed by a two-component flavin-dependent hydroxylase: The ActVA-ActVB sytem from Streptomyces coelicolor
Published on 1 December 2005
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Thesis presented December 01, 2005
Abstract:
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 a second component, the proper monooxygenase. Here, we study the two-component system ActVB and ActVA-Orf5, reported to be involved in tha last step of biosynthesis of the natural antibiotic actinorhodin in
Streptomyces coelicolor. ActVB has been already reported to be a NADH:flavin oxidoreductase able to catalyze the reduction of free flavin by NADH by a sequential ordered mechanism. Here, for the first time, we show that ActVA-Orf5 is an FMN-dependent monooxygenase which together with the help of ActVB, catalyzes the aromatic hydroxylation of its natural substrate DHK. The mechanism of the transfer of FMN between ActVB and ActVA-Orf5 has been investigated. Dissociation constant values for FMNox and FMNred with regard to ActVB and ActVA-Orf5 have been determined. The results clearly demonstrate that FMNred has a better affinity for ActVA than for ActVB whereas FMNox has a better affinity for ActVB than for ActVA. In full agreement with this finding, our data clearly indicate a thermodynamic transfer of FMNred from ActVB to ActVA-Orf5, without involving a particular interaction between the two protein components. In addition, we show that in the presence of FMNred and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic hydroperoxyflavin intermediate species. This intermediate reacts efficiently with the nucleophilic species DHK fo form its hydroxylated analogue: DHK-OH. In agreement with these data, we found that the electron rich hydroquinone form of DHK was an excellent substrate whereas the quinone was not. ActVA-ActVB system does not seem to be highly specific because the enantiomer of DHK, the NNM-A, as well as the lactonic analogue of NNM-A, the NNM-D, are also substrates in their hydroquinones forms. However, DHK is a much better substrate than NNM-A and NNM-D. Finally, the previously postulated product of the AvtVA-ActVB system, the antibiotic actinorhodin, was not formed in our experiments and the last step of its biosynthesis need to be reinvestigated.
Keywords:
Flavin reductase, monooxygenase flavin-dependent, C(4a)-hydroxyperoxyflavin, flavin transfer, aromatic hydroxylation, actinorhodin biosynthesis
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