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• W2023753940 abstract "The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction. The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction. The two-component flavin-dependent monooxygenases have recently emerged as an important class of enzyme systems involved in biological oxidations (1Galan B. Diaz E. Prieto M.A. Garcia J.L. J. Bacteriol. 2000; 182: 627-636Crossref PubMed Scopus (153) Google Scholar). They are composed of two enzymes. First, a NAD(P)H:flavin oxidoreductase that catalyzes the reduction of free flavin, flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), by reduced pyridine nucleotides, NADPH or NADH (2Kendrew S.G. Harding S.E. Hopwood D.A. Marsh E.N. J. Biol. Chem. 1995; 270: 17339-17343Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 3Parry R.J. Li W. J. Biol. Chem. 1997; 272: 23303-23311Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Kirchner U. Westphal A.H. Muller R. van Berkel W.J. J. Biol. Chem. 2003; 278: 47545-47553Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5Louie T.M. Xie X.S. Xum L. Biochemistry. 2003; 42: 7509-7517Crossref PubMed Scopus (64) Google Scholar, 6Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2003; 278: 296-303Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 7van den Heuvel R.H. Westphal A.H. Heck A.J. Walsh M.A. Rovida S. van Berkel W.J. Mattevi A. J. Biol. Chem. 2004; 279: 12860-12867Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Second, an oxygenase that binds the resulting free reduced flavin together with the substrate in the active site (8Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar, 9Gisi M.R. Xun L. J. Bacteriol. 2003; 185: 2786-2792Crossref PubMed Scopus (83) Google Scholar, 10Xun L. J. Bacteriol. 1996; 178: 2645-2649Crossref PubMed Google Scholar, 11Xun L. Webster C.M. J. Biol. Chem. 2004; 279: 6696-6700Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Beltrametti F. Marconi A.M. Bestetti G. Colombo C. Galli E. Ruzzi M. Zennaro E. Appl. Environ. Microbiol. 1997; 63: 2232-2239Crossref PubMed Google Scholar, 13Panke S. Witholt B. Schmid A. Wubbolts M.G. Appl. Environ. Microbiol. 1998; 64: 2032-2043Crossref PubMed Google Scholar, 14Blanc V. Lagneaux D. Didier P. Gil P. Lacroix P. Crouzet J. J. Bacteriol. 1995; 177: 5206-5214Crossref PubMed Scopus (71) Google Scholar, 15Thibaut D. Ratet N. Bisch D. Faucher D. Debussche L. Blanche F. J. Bacteriol. 1995; 177: 5199-5205Crossref PubMed Google Scholar, 16Bohuslavek J. Payne J.W. Liu Y. Bolton Jr., H. Xun L. Appl. Environ. Microbiol. 2001; 67: 688-695Crossref PubMed Scopus (56) Google Scholar, 17Xu Y. Mortimer M.W. Fisher T.S. Kahn M.L. Brockman F.J. Xun L. J. Bacteriol. 1997; 179: 1112-1116Crossref PubMed Google Scholar, 18Eichhorn E. van der Ploeg J.R. Leisinger T. J. Biol. Chem. 1999; 274: 26639-26646Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 19Xi L. Squires C.H. Monticello D.J. Childs J.D. Biochem. Biophys. Res. Commun. 1997; 230: 73-75Crossref PubMed Scopus (73) Google Scholar, 20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). There is general agreement that the reaction proceeds through an oxidation of the flavin by molecular oxygen to generate a flavin hydroperoxide intermediate (21Palfey B.A. Ballou D.P. Massey V. Valentine J.S. Foote C.S. Greenberg A. Liebman J.F. Active Oxygen in Biochemistry. Blackie Academic and Professional, Glasgow, Scotland, UK1995: 37-83Crossref Google Scholar). This is followed by transfer of a single oxygen atom from the electrophilic peroxide to the substrate, generating the oxidized product of the reaction (21Palfey B.A. Ballou D.P. Massey V. Valentine J.S. Foote C.S. Greenberg A. Liebman J.F. Active Oxygen in Biochemistry. Blackie Academic and Professional, Glasgow, Scotland, UK1995: 37-83Crossref Google Scholar). Therefore in this two-component system, NAD(P)H oxidation and the hydroxylation reaction are catalyzed by separate polypeptides. In all cases, with the exception of luciferase (22Jeffers C.E. Nichols J.C. Tu S.-C. Biochemistry. 2003; 42: 529-534Crossref PubMed Scopus (46) Google Scholar), there is no evidence for an interaction between the two components and a channeling mechanism allowing the flavin to travel from one protein to another within a protein complex. Accordingly, in most cases, the oxidoreductase component can be replaced by other flavin reductases, including the non-homologous ones from other organisms (4Kirchner U. Westphal A.H. Muller R. van Berkel W.J. J. Biol. Chem. 2003; 278: 47545-47553Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5Louie T.M. Xie X.S. Xum L. Biochemistry. 2003; 42: 7509-7517Crossref PubMed Scopus (64) Google Scholar). The flavin transfer process from the oxidoreductase to the oxygenase is thus simply under thermodynamic control (5Louie T.M. Xie X.S. Xum L. Biochemistry. 2003; 42: 7509-7517Crossref PubMed Scopus (64) Google Scholar, 20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The most extensively studied oxygenase components so far are those utilizing FADH2 as a cofactor, such as 4-hydroxyphenylacetate monooxygenase (HpaB) from Escherichia coli (8Xun L. Sandvik E.R. Appl. Environ. Microbiol. 2000; 66: 481-486Crossref PubMed Scopus (90) Google Scholar), phenol hydroxylase (PheA1) from Bacillus thermoglucosidaflurescens (4Kirchner U. Westphal A.H. Muller R. van Berkel W.J. J. Biol. Chem. 2003; 278: 47545-47553Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and styrene monooxygenase (StyA) from Pseudomonas fluorescens (12Beltrametti F. Marconi A.M. Bestetti G. Colombo C. Galli E. Ruzzi M. Zennaro E. Appl. Environ. Microbiol. 1997; 63: 2232-2239Crossref PubMed Google Scholar, 13Panke S. Witholt B. Schmid A. Wubbolts M.G. Appl. Environ. Microbiol. 1998; 64: 2032-2043Crossref PubMed Google Scholar). In contrast, the oxygenases utilizing FMNH2 (termed FMNred herein) have been much less investigated. Examples are those involved in the synthesis of the antibiotic pristinamycin in Streptomyces pristinaespiralis (14Blanc V. Lagneaux D. Didier P. Gil P. Lacroix P. Crouzet J. J. Bacteriol. 1995; 177: 5206-5214Crossref PubMed Scopus (71) Google Scholar, 15Thibaut D. Ratet N. Bisch D. Faucher D. Debussche L. Blanche F. J. Bacteriol. 1995; 177: 5199-5205Crossref PubMed Google Scholar), utilization of sulfur from aliphatic sulfonates in E. coli (18Eichhorn E. van der Ploeg J.R. Leisinger T. J. Biol. Chem. 1999; 274: 26639-26646Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), or desulfurization of fossil fuels by Rhodococcus species (19Xi L. Squires C.H. Monticello D.J. Childs J.D. Biochem. Biophys. Res. Commun. 1997; 230: 73-75Crossref PubMed Scopus (73) Google Scholar). In our laboratory, we have been investigating the FMN-dependent two-component enzyme system, consisting of ActVB (6Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2003; 278: 296-303Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the flavin reductase, and ActVA-Orf5 (termed ActVA herein) (20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the oxygenase, thought to participate in the last steps of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor (23Cole S.P. Rudd B.A. Hopwood D.A. Chang C.J. Floss H.G. J. Antibiot. (Tokyo). 1987; 40: 340-347Crossref PubMed Scopus (60) Google Scholar, 24Caballero J.L. Martinez E. Malpartida F. Hopwood D.A. Mol. Gen. Genet. 1991; 230: 401-412Crossref PubMed Scopus (74) Google Scholar, 25Fernandez-Moreno M.A. Martinez E. Boto L. Hopwood D.A. Malpartida F. J. Biol. Chem. 1992; 267: 19278-19290Abstract Full Text PDF PubMed Google Scholar) (Scheme 1). Our results have provided new insights into the mechanism of the reaction, especially regarding flavin reduction as well as flavin transfer from the oxidoreductase to the oxygenase (20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The reaction catalyzed by this enzyme system is particularly interesting and might have broader synthetic applications, and thus further studies of the mechanism, substrate specificity, and reaction efficiency are highly relevant. Here we report the results of our enzyme assays of the ActVB-ActVA system using the presumed natural substrate dihydrokalafungin (DHK) 3The abbreviations used are: DHKdihydrokalafunginRedreducedOxoxidizedNNM-Ananaomycin ANNM-Dnanaomycin DHPLChigh pressure liquid chromatographyMSmass spectrometry.3The abbreviations used are: DHKdihydrokalafunginRedreducedOxoxidizedNNM-Ananaomycin ANNM-Dnanaomycin DHPLChigh pressure liquid chromatographyMSmass spectrometry. (23Cole S.P. Rudd B.A. Hopwood D.A. Chang C.J. Floss H.G. J. Antibiot. (Tokyo). 1987; 40: 340-347Crossref PubMed Scopus (60) Google Scholar). Unexpectedly, we demonstrate that the DHK substrate appears to be utilized in its hydroquinone form and the only product formed is hydroxylated DHK. Although this enzyme system (or at least ActVB) was originally proposed to be involved in the dimerization to form actinorhodin (23Cole S.P. Rudd B.A. Hopwood D.A. Chang C.J. Floss H.G. J. Antibiot. (Tokyo). 1987; 40: 340-347Crossref PubMed Scopus (60) Google Scholar), we have not so far observed dimerization under the range of conditions used in our in vitro experiments. We also provide further evidence for the involvement of a FMN-OOH intermediate during the catalytic cycle, which is unusually stabilized by the active site of ActVA. dihydrokalafungin reduced oxidized nanaomycin A nanaomycin D high pressure liquid chromatography mass spectrometry. dihydrokalafungin reduced oxidized nanaomycin A nanaomycin D high pressure liquid chromatography mass spectrometry. FMN, NADH, and Nanaomycin A (NNM-A) were purchased from Sigma or Aldrich. Other reagent grade chemicals were obtained from Euromedex. Deazaflavin (5-deaza-5-carbariboflavin) was a gift from Dr. Philippe Simon (Grenoble, France). NNM-D, DHK, and actinorhodin were a gift from Professors E. N. G. Marsh, D. Hopwood, and S. Omura (to S. G. K.). Recombinants ActVA-Orf 5 and His-tagged ActVB were overexpressed in E. coli and purified as described in Refs. 6Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2003; 278: 296-303Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar and 20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar. Anaerobic experiments were performed at 18 °C in a Jacomex glove box equipped with a UV-visible cell coupled to an Uvikon XL spectrophotometer by optical fibers (Photonetics system). All the solutions were incubated anaerobically for 2 h before the beginning of each experiment. Deoxygenated stock solutions (500 μm) of FMN (ϵ445 nm = 12.5 mm–1 cm–1), NNM-A, NNM-D, and DHK were anaerobically photoreduced in the presence of 0.5 μm deazaflavin and 10 mm EDTA by irradiation for 30 min using a commercial slide projector placed at a distance of 3 cm. The reduced solutions were used within 1 day. With Reduced Pyronaphthoquinone Substrates—10 μm FMNred was first mixed in the anaerobic glove box with 10 μm reduced pyronaphthoquinone and 50 μm ActVA into a 100-μl air-tight spectrophotometric cuvette. The oxidation was initiated by the addition of 10 μl of pure oxygen-saturated water (1 mm O2) with a Hamilton syringe. The reaction was followed by UV-visible spectroscopy. At the end of the reaction, the mixture was immediately analyzed by HPLC-MS as described below. With Oxidized Pyronaphthoquinone Substrates—Various amounts of pure oxygen-saturated water (1 mm O2) were added to an air-tight microvial (150 μl) containing 100 μm oxidized pyronaphthoquinone (final concentration) to a final volume of 100 μl. Oxygen final concentrations ranging between 28 and 90 μm were obtained with this procedure. In the anaerobic glove box, 10 μl of this mixture was injected with a Hamilton syringe into an air-tight spectrophotometric cuvette containing 100 μl of 10–20 μm FMNred and 50 μm ActVA solution. The reaction was followed spectrophotometrically. At the end of the reaction, the mixture was analyzed and quantified by HPLC as described below. In the Absence of Pyronaphthoquinone Substrate—Under aerobic conditions, ActVB (155 nm) was added to a mixture containing 200 μm NADH, 46 or 80 μm FMN, 0 to 104 μm ActVA, and 20 mm Tris-HCl, pH 7.6, in a final volume of 100 μl. The reaction was monitored spectrophotometrically at 25 °C. In the Presence of Oxidized Pyronaphthoquinone Substrate—Under aerobic conditions, ActVB (155 nm) was added to a mixture containing 200 μm NADH, 4 μm FMN, 36 μm oxidized pyronaphthoquinone substrate (DHK or NNM-A), 0 to 180 μm ActVA in a 20 mm Tris-HCl, pH 7.6, buffer. The reaction was followed by UV-visible spectrophotometry at 25 °C. The appearance of the hydroxylated product was monitored at 520 nm and also analyzed by HPLC-MS as described below. Similar experiments were performed with successive additions of NADH (200 μm) to obtain a total hydroxylation of the pyronaphthoquinone substrates. The steady-state kinetic parameters of the nanaomycin A reductase activity of ActVB were determined in the anaerobic glove box at 18 °C, by monitoring the quinone reduction at 423 nm (ϵ423 nm = 4.0 mm–1 cm–1). Under standard conditions, the reaction mixture contained 200 μm NADH, 50 mm Tris-HCl, pH 7.6, and various amounts of NNM-A, in a final volume of 100 μl. The reaction was initiated by the addition of an ActVB solution containing 34 nm FMN (final concentration). Initial velocities were determined from the early linear part of the reaction progress curves and plotted as a function of NNM-A concentrations. Data were fitted according to the Michaelis-Menten equation using the Levenberg-Marquardt algorithm of Kaleidagraph®. 10 μm FMNred was mixed in an anaerobic glove box with 28 μm ActVA and 20 mm Tris-HCl, pH 7.6, buffer at 18 °C. A deoxygenated DHKox solution was then added rapidly with a Hamilton syringe to a final concentration of 10 μm and the reaction was monitored by UV-visible spectroscopy. HPLC analyses were performed with a 1100 Agilent chromatographic system coupled to a diode array UV-visible spectrophotometer. An aliquot fraction (100 μl) of the reaction mixture was diluted two times with an aqueous solution containing 0.1% trifluoroacetic acid prior to being loaded onto a C18 column (previously equilibrated with 65% of ultrapure water and 35% of acetonitrile, both containing 0.1% trifluoroacetic acid). Elution was conducted with a 35–100% acetonitrile (0.1% trifluoroacetic acid) linear gradient at a flow rate of 1 ml/min during 10 min and monitored by UV-visible spectrophotometry between 250 and 900 nm. Pyronaphthoquinone substrates and hydroxylated products were then quantified from the integration of the HPLC peak monitored at 423 and 520 nm, respectively. HPLC-tandem mass spectrometry analyses were performed with a 1100 Agilent chromatographic system coupled to an API 3000 triple quadripolar apparatus (Applied Biosystem/SCIEX) equipped with a turbo ionspray electrospray source used in the negative mode. Samples were loaded onto a 2 × 150-mm octadecylsilyl silica gel (5 μm particle size) column (Uptisphere, Interchim Montluçon, France) previously equilibrated with 65% ultrapure water containing 2 mm ammonium formate and 35% acetonitrile. Elution was carried out with a 35–100% acetonitrile linear gradient in 2 mm ammonium formate as the mobile phase, at a flow rate of 200 μl/min during 10 min. Mass analyses were performed in the multiple reaction monitoring mode. For this purpose, fragments corresponding to the decarboxylated ([M–44–H]) pseudomolecular ions were quantified. The transitions corresponding to pyronaphthoquinone, hydroxylated pyronaphthoquinone, and actinorhodin were monitored simultaneously with a dwell time set at 650 ms for each compound. The ActVA-ActVB System in the Absence of Pyronaphthoquinone Substrates: Evidence for a Flavin Hydroperoxide Intermediate under Steady-state Conditions—In the experiments described below, the complete flavin reductase (ActVB)-monooxygenase (ActVA) system was investigated in the absence of pyronaphthoquinone substrates under aerobic ([O2] = 200 μm) steady-state conditions. An excess of ActVA with regard to both ActVB and FMN was used to ensure that the reduced flavins (FMNred) produced by the action of ActVB/NADH were fully trapped within ActVA polypeptide chains (in agreement with the previously determined Kd values for FMNred with regard to ActVA and ActVB, 0.39 and 6.60 μm, respectively (20Valton J. Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2004; 279: 44362-44369Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar)). Furthermore, the FMN concentration was such that the ActVB enzyme was saturated (Km value for FMNox = 1 μm (6Filisetti L. Fontecave M. Nivière V. J. Biol. Chem. 2003; 278: 296-303Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar)). The reaction could be monitored by UV-visible spectroscopy because both NADH and FMN are characterized by specific light absorption bands in the 300–500-nm region. NADH (200 μm) oxidation was monitored at 340 nm, whereas FMNox reduction was followed at 445 nm. The results are shown in Fig. 1. During the first step of the aerobic incubation, we observed an oxidation of NADH (decrease of the intensity of the 340-nm band) that stopped (plateau of the intensity at 340 nm) after ∼100 s, when all NADH was consumed (Fig. 1A). During the first 100 s, FMNox was transformed (decrease of the intensity at 445 nm) almost quantitatively into a species whose concentration remained constant (plateau at 445 nm) as long as NADH was present in solution (Fig. 1A). During this steady-state, FMN was mostly in a state displaying a single absorption band at 370 nm in the UV-visible spectrum (the slight absorption above 450 nm corresponds to the residual FMNox form), characteristic for a flavin-hydroperoxide species (21Palfey B.A. Ballou D.P. Massey V. Valentine J.S. Foote C.S. Greenberg A. Liebman J.F. Active Oxygen in Biochemistry. Blackie Academic and Professional, Glasgow, Scotland, UK1995: 37-83Crossref Google Scholar), FMN-OOH (Fig. 1B, squares). After the 100-s reaction, when all NADH was oxidized, the flavin was slowly and totally converted back to FMNox, as shown from the increase of the intensity of the 445-nm absorption band (Fig. 1, A and B). From Fig. 1A, a first-order rate constant of k = 0.84 min–1 was determined for the decomposition of the FMN-OOH species. This process (100–359-s reaction time) occurred without any absorbing intermediate species, because spectral changes involved three isosbestic points at 333, 365, and 399 nm (Fig. 1B). Note that the first spectrum at 76 s does not share all these isosbestic points because it also contains absorption at 340 nm because of residual NADH. This experiment showed that, in the absence of substrate and under the reductive pressure of NADH, the intermediate FMN-OOH is present in aerobiosis under large steady-state concentrations and is thus spectrophotometrically observable. The same type of experiment was carried out with various concentrations of ActVA. As shown in Fig. 2A, the steady-state level of FMN-OOH, reflected by the plateau of the optical density at 445 nm (reaction time 50–100 s), increased with increased ActVA concentration. The amount of FMNox converted to FMN-OOH was determined from the A at 445 nm using the difference between its value at the end of the reaction (t > 350 s) and its value at the plateau (t = 50 s), assuming negligible contribution of the FMN-OOH species at this wavelength. It should be noted that in these conditions free FMNred does not accumulate because it is immediately oxidized by excess O2. Plotting this value as a function of ActVA concentration generates a straight line, indicating that FMN-OOH accumulation is directly proportional to the ActVA concentration. The slope in Fig. 2B gives the amount of FMN-OOH formed per ActVA molecule. The results demonstrated that under the experimental conditions used here, one molecule of FMN-OOH was formed per dimer of ActVA, at the steady state. The ActVA-ActVB System in the Presence of Pyronaphthoquinone Substrates: a Monooxygenation Reaction—The substrate of the enzyme system is presumed to be DHK (Schemes 1 and 2). However, enzymatic oxidation of DHK has not been shown in vitro. When DHK (36 μm) was incubated aerobically with the ActVA-ActVB system in the presence of 4 μm FMN and 200 μm NADH in 20 mm Tris-HCl buffer, pH 7.7, a product was formed at the expense of DHK, as shown by HPLC. No product could be obtained in the absence of ActVA (data not shown). Fig. 3A shows the chromatogram detected at the end of the reaction when all NADH has been oxidized (100 s). DHK is eluted at 7.4 min and the product at 7.7 min. The spectrum of the product is shown in Fig. 3B (triangles). The absorption band enjoys a large bathochromic shift with regard to that of DHK, from 423 to 507 nm, and is significantly different from that of actinorhodin. The ϵ values have been estimated as described below. Analysis of the new compound by LC-MS demonstrated that its mass differed from that of DHK by only one oxygen atom (m/z = 317 Da instead of 301 Da for DHK). This result showed that the product of the reaction in vitro was not actinorhodin (m/z = 633 Da), but rather a monooxygenated derivative of DHK, named DHK-OH in the following.FIGURE 3ActVA-ActVB hydroxylase activity: identification of the hydroxylated product by HPLC-MS and UV-visible spectroscopy. 4 μm FMNox was aerobically incubated with 50 μm ActVA, 155 nm ActVB, 36 μm DHK, 200 μm NADH in a 20 mm Tris-HCl buffer, pH 7.6, at 25 °C. A, the reaction mixture was analyzed after 100 s by HPLC-tandem mass spectrometry in the multiple reaction monitoring mode set up to analyze DHK and DHK-OH mass in a negative mode. B, UV-visible spectra of actinorhodin (○), DHK (□), and DHK-OH (▵). The ϵ values of the latter were determined from the steady-state experiments as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the experiment of Fig. 3, using 200 μm NADH, DHK (36 μm initial concentration) was only partially converted to DHK-OH, as monitored by HPLC. We thus carried out several reaction runs by repeated additions of 200 μm NADH and oxygen until all the DHK was oxidized to product. From one run to the following the spectrum did not change in shape and the area of the peak corresponding to DHK-OH increased by about the same extent to reach a plateau when all DHK was oxidized. Assuming that the only oxidation product under these conditions was DHK-OH and that conversion of DHK to DHK-OH was quantitative, one thus could determine the extinction coefficient for DHK-OH at 507 nm, with a value of 4.4 mm–1 cm–1 (as compared with 0.27 mm–1 cm–1 for DHK). This extinction coefficient of the band at 520 nm for DHK-OH was comparable with that of the band at 423 nm for DHK. This was also observed for quinone analogs such as 5-hydroxy-1,4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone, which display visible spectra remarkably similar to those of DHK and DHK-OH, respectively (26Khan M.S. Khan Z.H. Spectrochimica Acta Part A Mol. Spectrosc. 2005; 61: 777-790Crossref PubMed Scopus (41) Google Scholar). It was clear that the reaction yield, calculated as the amount of DHK-OH divided by the amount of consumed NADH (200 μm) during the first run, was low. This yield increased with increased ActVA concentration but leveled off at about 10% with the highest concentrations (Fig. 4). The dependence of the yield on FMN concentration was also studied and showed that the optimal concentration of FMN was 4 μm, using 37 μm ActVA (data not shown). The same complete study, involving product characterization (UV-visible spectroscopy and mass spectrometry) and calculation of reaction yields, was carried out with NNM-A as a substrate (data not shown). NNM-A is the enantiomer of DHK (Scheme 2). As for DHK, only one monooxygenated product was obtained. Fig. 4 shows the dependence of the yield of NNM-A oxygenation upon ActVA concentration, demonstrating that it is a poorer substrate. Taken together, these results indicated a very inefficient coupling of ActVB (flavin reduction) and ActVA (monooxygenation) activities. A possible source of uncoupling was the unproductive reaction of free FMNred with molecular oxygen during flavin transfer from ActVB to ActVA. However, as a large excess of ActVA was used in our experiments to ensure fast and complete binding of FMNred, this factor is unlikely to be a significant contribution to the uncoupling. Another possible source of uncoupling was identified as described below. Quinone Reductase Activity of the ActVA-ActVB System—Reduced flavins are excellent reducing agents with regard to a variety of electron acceptors such as oxygen, ferric complexes, and also quinones (27Gaudu P. Touati D. Nivière V. Fontecave M. J. Biol. Chem. 1994; 269: 8182-8188Abstract Full Text PDF PubMed Google Scholar, 28Fontecave M. Eliasson R. Reichard P. J. Biol. Chem. 1987; 262: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 29Filisetti L. Valton J. Fontecave M. Nivière V. FEBS Lett. 2005; 579: 2817-2820Crossref PubMed Scopus (14) Google Scholar). We thus suspected the quinone compounds (DHK and NNM-A) used as substrates of the ActVA-ActVB system to be reduced by the FMNred generated by ActVB. Indeed, free FMNred, obtained through ActVB flavin reductase activity or through anaerobic irradiation in the presence of deazaflavin, was shown to quantitatively reduce NNM-A, as shown from the intensity of the final band at 353 nm, characteristic of its hydroquinone form (data not shown). We also investigated the quinone reductase activity of an ActVB preparation containing a tightly bound FMN (29Filisetti L. Valton J. Fontecave M. Nivière V. FEBS Lett. 2005; 579: 2817-2820Crossref PubMed Scopus (14) Google Scholar) in the absence of free flavin. To avoid competition with molecular oxygen, all the experiments were carried out within an anaerobic glove box. When 200 μm NADH was incubated with ActVB containing 34 nm FMN bound, in Tris buffer, pH 7.6, no oxidation of NADH could be detected. Addition of a quinone substrate such as NNM-A in this medium resulted in its fast reduction by NADH, as shown by the decrease of the intensity of the 423 nm absorption band characteristic for the quinone. Fig. 5A shows the initial and final UV-visible spectrum in an experiment with NADH (200 μm) in excess with regard to the quinone (130 μm). At the end of the reaction, there is no more quinone as shown from the lack of the band at 423 nm. The absorption band at around 350 nm is a mixture of a band at 340 nm (residual NADH) and a new band at 353 nm, characteristic of the corresponding NNM-A hydroquinone (30Tanaka H. Minami-Kakinuma S. Omura S. J. Antibiot. (Tokyo). 1982; 35: 1565-1570Crossref PubMed Scopus (9) Google Scholar). The contribution of the flavin i" @default.
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• W2023753940 creator A5043101051 @default.
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