The flavin reductase ActVB from Streptomyces coelicolor: characterization of the electron transferase activity of the flavoprotein form.

International audienceThe flavin reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the flavin reductase activity. By studying the ferric reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide

A two-component flavin-dependent monooxygenase involved in actinorhodin biosynthesis in Streptomyces coelicolor.

International audienceThe 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

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site.

International audienceSuperoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR

Non-specific protein-DNA interactions control I-CreI target binding and cleavage

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications

Neuromatch Academy: a 3-week, online summer school in computational neuroscience

Neuromatch Academy (https://academy.neuromatch.io; (van Viegen et al., 2021)) was designed as an online summer school to cover the basics of computational neuroscience in three weeks. The materials cover dominant and emerging computational neuroscience tools, how they complement one another, and specifically focus on how they can help us to better understand how the brain functions. An original component of the materials is its focus on modeling choices, i.e. how do we choose the right approach, how do we build models, and how can we evaluate models to determine if they provide real (meaningful) insight. This meta-modeling component of the instructional materials asks what questions can be answered by different techniques, and how to apply them meaningfully to get insight about brain function

Réaction d'hydroxylation aromatique catalysée par une hydroxylase flavine-dépendante à deux composants : le système ActVA-ActVB de Streptomyces coelicolor

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. Here, we study the two-component system ActVB and ActVA-OrF5, reported to be involved in the 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, catalyze 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 to 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 ActVA-ActVB system, the antibiotic actinorhodin, was not formed in our experiments and the last step of its biosynthesis need to be reinvestigated.Il y a une dizaine d'années, de nouvelles flavoenzymes nommées hydroxylases flavine-dépendantes à deux composants ont été identifiées chez certains microorganismes. Le rôle physiologique de ces enzymes est maintenant bien connu. Elles sont impliquées dans les processus de biosynthèse et de biodégradation d'une multitude de molécules organiques. Ces hydroxylases sont composées de deux enzymes distinctes. La première est une flavine réductase qui catalyse la formation de flavine réduite nécessaire au fonctionnement de la seconde enzyme, une monooxygénase flavine-dépendante. Au début de notre projet, le mécanisme enzymatique de ces nouvelles hydroxylases était encore inconnu. Pour comprendre le détail de leur fonctionnement, nous avons choisi d'étudier le système ActVA-ActVB, un nouveau membre de la famille des hydroxylases flavine-dépendantes impliqué dans la dernière étape de biosynthèse de l'actinorhodine, un antibiotique naturel synthétisé par Streptomyces coelicolor. La caractérisation préalable de ActVB avait permis de montrer que cette enzyme était une NADH:FMN oxydoréductase capable de catalyser la réduction du FMN par le NADH selon un mécanisme de type séquentiel ordonné. Nos résultats ont permis d'identifier ActVA-Orf5, une monooxygénase flavine-dépendante capable d'utiliser la flavine réduite fournie par ActVB pour catalyser l'hydroxylation du précurseur de l'actinorhodine, la DHK. Le mécanisme de transfert de flavine entre les deux protéines a été étudié. Pour cela, les constantes de dissociation du FMNox et FMNred vis-à-vis de ActVA et ActVB ont été déterminées. Nos donnés montrent clairement qu'à l'état réduit, la flavine est bien plus affine pour la monooxygénase ActVA que pour la réductase ActVB alors qu'à l'état oxydé, elle possède une meilleure affinité pour la réductase que pour la monooxygénase. Cette différence d'affinité permet d'orienter le transfert de flavine d'une protéine à l'autre sans nécessiter d'interaction entre les deux protéines. Nous avons montré de plus que ActVA avait la capacité de stabiliser un intermédiaire activé de l'oxygène, une espèce électrophile nommée C(4a)-hydroperoxyflavine, au sein de son site actif. Cet intermédiaire réagit très rapidement avec la DHK nucléophile pour former son analogue hydroxylé : la DHK-OH. En accord avec ce mécanisme, il semble que le pouvoir nucléophile du substrat est très important pour cette réaction car seule la forme réduite à deux électrons de DHK (hydroquinone) est hydroxylée. D'autre part, ActVA ne semble pas être très spécifique car elle parvient également à catalyser l'hydroxylation de l'énantiomère de la DHK, la NNM-A et de l'analogue lactonique de la NNM-A, la NNM-D. Finalement le système ActVA-ActVB n'a pas la capacité de dimériser la DHK-OH pour former l'actinorhodine et l'enzyme intervenant dans la dernière étape de cette biosynthèse reste à identifier