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J. Biol. Chem., Vol. 279, Issue 43, 44362-44369, October 22, 2004

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A Two-component Flavin-dependent Monooxygenase Involved in Actinorhodin Biosynthesis in Streptomyces coelicolor^*

Julien Valton, Laurent Filisetti, Marc Fontecave, and Vincent Nivière

From the Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DRDC-CEA/CNRS/UniversitéJoseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble Cedex 9, France

Received for publication, July 9, 2004 , and in revised form, August 3, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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. 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 (FMN_ox and FMN_red) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMN_red 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 FMN_ox binds to ActVB, where it is reduced, and the resulting FMN_red 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
There is a great variety of monooxygenases catalyzing oxygen activation and oxygen transfer reactions in the living world. The most extensively studied ones are the cytochrome P450-dependent monooxygenases (1) and the enzymes belonging to the methane monooxygenase family (2), both using an iron center for O₂ activation. Also physiologically important are the flavin-dependent monooxygenases. These systems can be divided into two groups. The first one consists in enzymes with FAD or FMN as prosthetic groups, in which reduction of the flavin by reduced pyridine nucleotides, NADH or NADPH, and oxidation of substrates are carried out on a single polypeptide chain (3). A second class of enzymes that utilize the flavin, FAD or FMN, as a cosubstrate rather than a prosthetic group recently emerged. A separate NAD(P)H:flavin oxidoreductase is, thus, required to supply reduced flavins that bind to the oxygenase component, where they activate O₂ for substrate oxidation (Scheme 1) (4). Therefore, in this two-component system, NAD(P)H oxidation and the hydroxylation reaction are catalyzed by separate polypeptides. For years, the only prototype for this class of monooxygenases was the luciferase enzyme, which has been the subject of numerous studies (3, 5, 6). However, it is only recently that the importance of such systems in oxidative metabolism, in particular within microorganisms, has been realized.

View larger version (12K): [in this window] [in a new window]   SCHEME 1

The monooxygenase components can be further divided into two groups on the basis of the type of the flavin used in the reaction. The first group consists in FADH₂-utilizing monooxygenases, which are represented by several well characterized systems such as 4-hydroxyphenylacetate 3-monooxygenase (HpaB) of Escherichia coli (13) and 2,4,5-trichlorophenol monooxygenase from Burkholderia cepacia (14, 15). 2,4,6-Trichlorophenol monooxygenase (TcpA) from Ralstonia eutropha (16), phenol hydroxylase (Phe A1) from Bacillus thermoglucosidasius (9), and styrene monooxygenase (StyA) from Pseudomonas flurescens ST (17, 18) belong to that family but have not been characterized yet. The second group consists in FMNH₂-utilizing monooxygenases, which have been much less investigated in terms of their structure, mechanism, and substrate specificity. Examples of such FMN-dependent systems are those involved in the synthesis of the antibiotic pristinamycin in Streptomyces pristinaespiralis (19, 20), biodegradation of polyaminocarboxylates such as EDTA (21) or nitrilotriacetic acid (NTA)¹ (22) by microorganisms, utilization of sulfur from aliphatic sulfonates (23), and desulfurization of fossil fuels by Rhodococcus species (24).

The results reported here concern the enzyme system we have selected to study the chemistry of FMN-utilizing monooxygenases. The system participates in the last step of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor (Scheme 2), which consists of the hydroxylation of the precursor dihydrokalafungin and the coupling of two molecules of the hydroxylated product (25–27).

View larger version (8K): [in this window] [in a new window]   SCHEME 2

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—FMN, NADH, 1,5-dihydroanthraquinone (DHAQ) were purchased from Sigma or Aldrich. Other reagent-grade chemicals were obtained from Euromedex. Deazaflavin (5-Deaza-5-carbariboflavin) was a gift from Philippe Simon (Grenoble, France).

Construction of ActVA-ORF5 Expression Plasmid—Plasmid pIJ2305 containing the whole actV region (28) was isolated from S. coelicolor with the Plasmid Midiprep kit (Qiagen). ActVA-ORF5 gene was amplified by PCR from pIJ2305 using the primer 5'-GGAATTCCATATGAGCGAGGACACGATGACC-3' (EcoR1 and NdeI restriction sites are underlined) and the reverse primer 5'-CGCGGATCCTCAGCCATCGTTGCTCCTCCT-3' (the BamH1 restriction site is underlined). The PCR product was digested by EcoR1-BamH1, and the resulting fragment was ligated into pUC18 (pUC18-ActVA). This plasmid was sequenced to confirm that no changes had been introduced in the actVA-ORF5 sequence during PCR. The NdeI-BamH1 fragment derived from pUC18-ActVA was subsequently cloned into pT7-7 at the same restriction sites, resulting in the plasmid named pActVA used for the overexpression of the actVA-ORF5 gene product.

Overexpression and Purification of ActVA-ORF5—E. coli BL21 (DE3) pLysS harboring pActVA was grown at 37 °C and 220 rpm in a 3-liter flask containing 1 liter of Luria and Bertani medium in the presence of 200 µg/ml ampicillin and 34 µg/ml chloramphenicol. Expression of ActVA-ORF5 was induced by adding isopropyl-1-thio--D-galactopyranoside to a final concentration of 380 µM when an A_{600 nm} = 0.4 was reached. After 5–6 h of growth, cells were harvested by centrifugation at 4 °C and suspended in 10 mM Tris-HCl, pH 7.6, 10% glycerol (buffer A). All the following steps except cell disruption were performed at 4 °C. Extraction of soluble proteins was performed by lysozyme treatment (0.5 mg/ml lysozyme, incubation time 45 min at room temperature) followed by French press disruption. The resulting cell extract was clarified by ultracentifugation at 45,000 rpm for 90 min (rotor TI 50.2 Beckman), and the supernatant was used as crude extract for purification. Crude extract (700 mg in proteins) was loaded onto a 60-ml Q-Sepharose XL column (Amersham Biosciences) previously equilibrated with buffer A. The column was then washed with 60 ml of buffer A plus 25 mM NaCl, and the proteins were eluted with a linear 0–500 mM NaCl gradient. Fractions containing ActVA, eluted with 120 mM NaCl, were pooled and concentrated to 2 ml by ultrafiltration using a 10,000 M_r regenerated cellulose membrane (Amicon). 30% of the total protein fraction was lost in this step, probably due to irreversible adsorption to the membrane. The concentrated fraction was loaded onto a Superdex 200 gel filtration column (120 ml, from Amersham Biosciences) previously equilibrated with buffer A and then eluted with the same buffer. Fractions containing pure ActVA, about 95% as determined by SDS-PAGE, were pooled, concentrated as described above, and stored at –80 °C.

Preparation of ApoActVB—Overexpression and purification of His-tagged ActVB was carried out as previously reported (10). All the following steps were performed at 4 °C. Purified His-tagged ActVB (2 mg) was loaded onto a 1 ml Ni-NTA column (Amersham Biosciences) and treated with 1.5 column volumes of 2 M KBr and 2 M urea at acidic pH (29). Renaturation on the column was achieved with 4 column volumes of 50 mM Tris-HCl, pH 7.6, 10% glycerol, and the protein was recovered with 3 column volumes of 500 mM imidazole, 25 mM Tris-HCl, pH 7.6, 10% glycerol. Because apoActVB was unstable in the presence of imidazole, the protein solution was immediately loaded onto a Nap 10 column and eluted with 25 mM Tris-HCl, pH 7.6, 10% glycerol. The yield of the overall process was about 40%.

Analytical Determinations—The native molecular mass of ActVA was determined with an analytical Superdex 200 gel filtration (Amersham Biosciences) equilibrated with 10 mM Tris-HCl, pH 7.6, containing 150 mM NaCl. Bovine serum albumin (66 kDa), ovalbumin (45 kDa), chymotrypsin (20.1 kDa), and cytochrome c (12.4 kDa) were used as markers of molecular mass. The void volume was determined with ferritin (450 kDa). Protein concentration was determined using the Bio-Rad protein assay reagent with bovine serum albumin as a standard.

Flavin Binding to ActVA-ORF5 and ActVB—UV-visible absorbance spectra were recorded with a Hewlett-Packard 8453 photodiode array instrument at 18 °C. K_d values of FMN_ox for ActVB and ActVA were calculated from the variation of the absorbance values at the selected wavelengths using Equation 1,

(Eq. 1)

where A_o is the initial absorbance, A_x is the absorbance after a given addition of the protein, and A_f is the absorbance at the end of the titration. Making the hypothesis that ActVA and ActVB contain one binding site per monomer, [FMN_bound] and [protein_free] were calculated from the total concentrations of FMN ([FMN_tot]) and protein ([protein_tot]) using Equations 2 and 3.

(Eq. 2)

(Eq. 3)

ActVA fluorescence emission spectra were recorded at 18 °C with a Jasco FP 6500 spectrofluorimeter using a 1-cm square cuvette (Helma). The excitation wavelength was set to 295 nm. The measured fluorescence spectra were corrected from inner filter effects due to FMN_ox or FMN_red absorption during excitation as well as during emission. The corrected fluorescence (F_corr) was calculated according to the method described in (30) using Equation 4,

(Eq. 4)

where L is the cuvette full path length (1 cm), [FMN] the total concentration of FMN, _ex is the extinction coefficient of FMN at excitation wavelength (295 nm), and _em is the extinction coefficient of FMN for each emission wavelength between 300 and 400 nm. Because the inner filter effect is not homogenous in the entire cuvette, we divided it into 100 identical points and defined their position by x and y coordinates, making the hypothesis that their surfaces are infinitely small. For each position and each wavelength between 295 and 400 nm, the factors representing the inner filter effect were calculated and were subsequently summed to determine an F corrected (F_corr) value. F_corr precisely corresponds to the intrinsic protein fluorescence. K_d values were calculated from the variation of the corrected fluorescence intensity at 338 nm (F_corr) using Equations 1, 2, and 3.

Anaerobic Experiments—Anaerobic experiments were performed in a Jacomex glove box equipped with a UV-visible cell coupled to a Uvikon XL spectrophotometer by optical fibers (Photonetics system). All the solutions were incubated anaerobically 2 h before the beginning of each experiment. For FMN reduction, an FMN stock solution (500 µM) was anaerobically and quantitatively photoreduced by 30 min of irradiation with a commercial slide projector placed at a distance of 3 cm in the presence of 2 µM deazaflavin and 10 mM EDTA. For oxidation of reduced flavin, as a standard procedure aerated water was prepared for each experiment by bubbling 100% oxygen gas for 30 min into an air-tight Eppendorf tube containing 1 ml of water. FMN_red oxidation was performed in the anaerobic glove box by injecting 5 µl of the oxygen-saturated water (1 mM) into a 100-µl air-tight spectrophotometric cuvette with an Hamilton syringe.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ActVA-ORF5 as the Potential Flavin-dependent Hydroxylase—Among the six genes present in the actVA region (26), only ORF5 displays sequence homologies with flavin-dependent hydroxylases. This becomes particularly obvious when the sequence of the corresponding protein is compared with the product of the tdsC gene from Paenibacillus spA11–2 (31) and the product of the pheA gene (32) (Supplemental Fig. 1). TdsC is an FMN-utilizing monooxygenase associated with TdsD, a NADH:FMN oxidoreductase, and involved in the oxidation of dibenzothiophene (31). A similar system is found in Rhodococcus erytropolis with DszC acting as a FMN-utilizing monooxygenase and DszD as a NADH:FMN oxidoreductase displaying strong sequence homologies with ActVB (24). Therefore, this analysis suggests that actVA-ORF5 gene product might be the monooxygenase associated with ActVB for the last step of actinorhodin biosynthesis.

Purification of ActVA-ORF5—The details of the methods used to clone and express the actVA-ORF5 gene in E. coli and purify the corresponding protein are given in the experimental section. Briefly, E. coli Bl 21 (DE3) pLysS strain was transformed with the expression vector pActVA-ORF5 derived from pT7-7 containing the actVA-ORF5 gene. Bacterial soluble extracts were subjected to an anion exchange chromatography, and the enriched fractions, as judged by SDS-PAGE analysis, were further purified by gel filtration on a Superdex 200 column. The protein was obtained 95% pure as determined by SDS-PAGE analysis (data not shown). No proteolysis occurred all through the purification process (data not shown). Determination of its N-terminal sequence (SEDTHT) confirmed that the purified protein was indeed the product of the actVA-ORF5 gene. For the sake of simplicity, in the following this protein is named ActVA. Analysis by mass spectroscopy showed that the protein, with a molecular mass of 39,715 Da, lacked the N-terminal methionine. Chromatography of pure ActVA on a calibrated Superdex 200 gel filtration column showed that the protein was dimeric in solution, with an experimental mass of about 77,000 Da (data not shown). Finally, no evidence for a protein-bound chromophore could be obtained by UV-visible spectroscopy.

At room temperature the protein spontaneously precipitated in the presence of 250 mM NaCl. Thus, the following experiments using pure ActVA were carried out in 50 mM Tris-HCl, pH 7.6, buffer in the absence of additional salt.

ActVA-ORF5, an Active Monooxygenase System—The precursor of actinorhodin is dihydrokalafungin. However, this compound is not commercially available and difficult to obtain either from natural sources (25) or by chemical synthesis (33). Thus, we used 1,5-dihydroanthraquinone (DHAQ) as a substrate analog to check whether the ActVA protein was indeed able to catalyze an hydroxylation reaction in combination with ActVB. The assay mixture under aerobic conditions contained 200 µM NADH, 5 µM FMN, 50 µM DHAQ, 11 nM ActVB, and various amounts of ActVA in 2 ml of 50 mM Tris-HCl, pH 7.6. After 1 h at 30 °C, DHAQ and its oxidation products were extracted with MeOH and analyzed by electrospray ionizationmass spectrometry. As shown in Fig. 1, a peak at m/z = 255 was observed besides the peak at m/z = 239 corresponding to DHAQ. This proved the formation of a product corresponding to DHAQ plus one oxygen atom (+16), thus deriving from DHAQ by monooxygenation. This peak was absent from the mass spectrum when one of the following components of the system was excluded from the reaction mixture: FMN, NADH, ActVB or DHAQ (data not shown). In addition, a control reaction carried out within an anaerobic glove box did not result in the production of the oxygenated product either (data not shown). In conclusion, taken together, these preliminary experiments reported here clearly indicate that the product of the actVAORF5 gene is a flavin-dependent monooxygenase.

View larger version (16K): [in this window] [in a new window]   FIG. 1. ActVA-ActVB hydroxylase activity. ActVB (11 nM) and ActVA (5 µM) were aerobically incubated with FMNox (5 µM), NADH (200 µM), and DHAQ (1,5-dihydroanthraquinone) (50 µM) in 50 mM Tris-HCl, pH 7.6, for 1 h at 30 °C. Products were then extracted with methanol and analyzed by electrospray-mass spectrometry. The electrospray mass spectrum of the organic extract and the structure of DHAQ are shown (m/z = 239).

As reported previously, a significant proportion (20–30%) of the purified ActVB polypeptides contained one bound FMN (10). Removal of FMN for the production of ActVB in the pure apoprotein form, named apoActVB, was thus achieved first before the binding experiments. The use of the His-tagged form of ActVB bound to a Ni-NTA column allowed a fast and efficient procedure for the preparation of apoActVB, as described under "Experimental Procedures." The protein bound to the Ni-NTA column was treated first with a solution containing 2 M urea and 2 M KBr. This was followed by renaturation, elution with imidazole, and desalting. The protein obtained from that process retained its ability to bind FMN, as judged from the appearance during incubation of apoActVB with FMN, of an absorption band centered to 457 nm, characteristic of the ActVB·FMN complex (10). ApoActVB was shown to be active as a flavin reductase in a standard assay using NADH and riboflavin or FMN as substrates, with K_m values for flavins (data not shown) comparable with those obtained with the as-isolated enzyme (10). Taken together, these data showed that apoActVB was fully active, well folded, and could, thus, be used in our following investigations.

Fig. 2 shows the UV-visible spectrum of the oxidized form of FMN (FMN_ox) under three different conditions. The _max value of the low energy visible band is 445 nm for free FMN_ox, 438 nm for FMN_ox in the presence of an excess of ActVA, and 457 nm for FMN_ox in the presence of an excess of ActVB. This showed that FMN_ox bound to ActVB, as previously demonstrated (10) but also to ActVA. Therefore, the _max value could be used as an indicator for whether FMN_ox is free or bound to ActVA or ActVB.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Light absorption spectra of FMNox. 10 µM FMNox was dissolved in 10 mM Tris-HCl, pH 7.6, alone (), in the presence of 200 µM ActVA (), or 40 µM ActVB (). OD, optical density.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Spectrophotometric titration of FMNox with ActVA. 10 µM FMNox in 50 mM Tris-HCl, pH 7.6, was titrated with ActVA. Bottom to top, concentrations of protein used were 0, 12, 34, 72, and 91 µM. The inset shows a plot of bound FMNox versus concentration of free ActVA, determined from the fractional absorbance change at 440 nm. A Kd value of 19.4 ± 6.3 µM was determined. OD, optical density.

Because FMN_red does not exhibit marked absorbance bands, K_d values for FMN_red could not be obtained by the same spectrophotometric titration experiments. For that purpose, we took advantage of specific spectroscopic properties of FMN_red as described in the following.

In the case of ActVB binding of FMN_red in its active site in the presence of NAD⁺ results in the formation of a broad absorption band between 550 and 800 nm that is characteristic for a charge transfer complex between NAD⁺ and FMN_red (Ref. 10 and Fig. 4). Such a complex is also formed as the result of the oxidation of NADH by FMN_ox in the active site of ActVB. As shown in Fig. 4, the intensity of the charge transfer band increased upon the successive addition of FMN_red in the presence of an excess of NAD⁺. A plot of the bound ActVB versus free FMN_red determined from the variation of the absorbance of the charge transfer band at 680 nm gave a K_d value of 6.6 ± 0.6 µM for FMN_red with regard to the [ActVB·NAD⁺] complex (inset in Fig. 4).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Spectrophotometric titration of ActVB with FMNred in the presence NAD+. ApoActVB (24 µM) was incubated in anaerobiosis with increasing amounts of FMNred in the presence of 2 mM NAD+ in 50 mM Tris-HCl, pH 7.6, at 18 °C. Bottom to top, 500–800 nm absorption spectra after the addition of 0, 10, 18, 37, 53, and 106 µM FMNred. The inset shows a plot of bound ActVB as a function of free FMNred, determined from the fractional absorbance change at 680 nm. A Kd value of 6.6 ± 0.6 µM was determined. OD, optical density.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Fluorimetric titration of ActVA with FMNox. ActVA (37 µM) was titrated by different concentrations of FMNox in 50 mM Tris-HCl, pH 7.6. Top to bottom, ActVA fluorescence spectra (excitation at 295 nm) after the addition of 0, 12, 36, 85, and 360 µM FMNox. Inner filter effects due to the absorption of FMNox at 295 nm and between 300–420 nm were corrected (see "Experimental Procedures"). The inset shows a plot of bound ActVA as a function of free FMNox, determined from the fluorescence intensity at 339 nm. A Kd value of 26.3 ± 3.2 µM was determined.

Table I summarizes the K_d values obtained here for both FMN_ox and FMN_red, characterizing their interaction with apo-ActVA and apoActVB. We deduce from these K_d values that FMN transfer is thermodynamically favorable from ActVA to ActVB in the oxidized state and from ActVB to ActVA in the reduced state.

View larger version (14K): [in this window] [in a new window]   FIG. 6. FMNox transfer from ActVA to ActVB. ActVA (140 µM) was mixed with FMNox (10 µM) in 50 mM Tris-HCl, pH 7.6. Increasing amounts of apoActVB were added, and the maximal absorption wavelengths were determined and plotted as a function of the ActVB/ActVA ratio.

Oxidation of FMNH₂; Evidence for a Flavin-Peroxide Intermediate—As shown above, FMN_red is a good ligand for ActVA. In the following, the study of the reaction of the ActVA·FMN_red complex with molecular oxygen is reported. In the experiment shown in Fig. 7, 16 µM FMN_red was anaerobically incubated with 140 µM ActVA, so that the flavin was totally complexed to the protein (Table I). The addition of oxygen-saturated water (50 µM O₂ final concentration) resulted in the complete oxidation of the flavin within 5 min as shown by light absorption spectroscopy (Fig. 7). The final spectrum with two bands at 370 and 438 nm was superimposable to that of a preparation of FMN_ox-containing ActVA. Therefore, it showed that the flavin stayed in the protein during the whole oxidation reaction. In the inset of Fig. 7, absorbances at 380 and 438 nm are reported as the function of time after the addition of O₂. At 380 nm, the kinetic trace indicated that the reaction with O₂ proceeded in two steps; that is, an initial fast phase occurring during mixing followed by a second slower phase. At 438 nm, only one phase was observed without evidence for the first rapid event observed at 380 nm. Without taking into account the initial fast phase at 380 nm, both kinetics at 380 and 438 nm were found exponential, with the same apparent first-order rate constant value, k = 0.44 ± 0.02 min^–1. Moreover, this rate constant was not a function of O₂ concentration (data not shown). This process was found to be much slower than the oxidation of free FMN_red under the same conditions (data not shown), suggesting that ActVA binds FMN_red in an environment that protects it significantly from oxidation.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Reaction of ActVA·FMNred complex with molecular oxygen. FMNred (16 µM) was incubated anaerobically with an excess of ActVA (140 µM) in 10 mM Tris-HCl, pH 7.6. The reaction was initiated by the addition of 50 µM O2. Bottom to top, spectra were recorded at 15, 82, 198, and 740 s. The inset shows the variation of absorbance at 380 nm () and 438 nm () as a function of time. Lines were calculated for best fit to a exponential model. OD, optical density.

Oxidation of FMN_red in complex with ActVA is also a very slow process when compared with that of FMN_red in complex with ActVB. This was shown in the experiment described below. ActVB·FMN_ox complex was treated anaerobically with a slight excess of NADH to generate ActVB·FMN_red. Oxidation of the flavin was initiated by the addition of aerated water and monitored by UV-visible spectroscopy. Fig. 8 shows the initial oxidation rates V_i for the O₂-dependent oxidation of ActVB·FMN_red in the presence of increasing amounts of ActVA. The observation that V_i decreased with increased ActVA was consistent with the notion that FMN_red spontaneously moved from ActVB to ActVA and was oxidized more slowly within the latter than within ActVB. As shown in Fig. 8, in the presence of 1 eq of ActVA compared with ActVB, the oxidation rate was similar to that of ActVA·FMN_red alone. This is in agreement with the much better affinity of FMN_red for ActVA than for ActVB. In addition, formation of FMN_ox within ActVB occurred with no observable intermediate species absorbing at 380 nm (data not shown). Therefore, a slow oxidation process via a FMN-OOH species is specific for ActVA.

View larger version (11K): [in this window] [in a new window]   FIG. 8. Effect of ActVA on the initial velocity of ActVB·FMNred complex oxidation. FMNox (16 µM) in the presence of apoActVB (50 µM) was reduced with 1 eq of NADH in 10 mM Tris-HCl, pH 7.6. Then the mixture was anaerobically incubated with different amounts of ActVA. O2 (50 µM final concentration) was rapidly added, and the initial velocity of FMNred oxidation was calculated from the increase of the absorbance at 457 nm as a function of time.

Interaction of ActVA with ActVB—To verify that ActVA and ActVB might not form a complex in solution, the following experiment was carried out. When an equimolar mixture of His-tagged ActVB and ActVA was loaded onto the Ni-NTA column, ActVA was totally recovered in the run-through fraction and could not be detected in the imidazole fraction containing ActVB (data not shown). These data suggest that under the conditions of the experiment, no interaction between ActVA and ActVB has been observed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The two-component flavin-dependent monoxygenases seem to be widely distributed within the bacterial world and used in a large variety of biosynthetic and metabolic reactions. However, whereas the flavin reductase component of these systems, which provides reduced flavins required for O₂ activation, has been the subject of detailed mechanistic and structural studies, very little is known on the monooxygenase component, where the oxidation takes place.

Our investigation of the final step of actinorhodin synthesis, which consists in the conversion of dihydrokalafungin into actinorhodin (Scheme 2) led us to characterize such a two-component FMN-dependent monooxygenase system. We first reported the purification and the characterization of ActVB, the NADH:FMN oxidoreductase associated with the reaction (10). The oxidation reaction itself is not a trivial reaction since it involves two oxidation steps, a dimerization and an aromatic hydroxylation. Not only is the order of the two steps still unknown, but it is also not sure whether there are different genes for each step.

The ActVA region of the act cluster carries several genes involved in actinorhodin biosynthesis (26). Because of the general difficulties in purifying the enzymes of that region coupled with the lack of commercial substrates required for biochemical studies, so far only ORF6 was assigned to a defined function, catalysis of the conversion of 6-deoxydihydrokalafungin to dihydrokalafungin (34, 35). However, we speculated that the ORF5 product, named ActVA here for sake of simplicity, could be the enzyme partner of ActVB for catalyzing one or the two steps of the oxidation of dihydrokalafungin to actinorhodin for the following reasons. First, a DNA fragment containing the actVA-ORF5 region could cause Streptomyces sp. AM 7161, the producer of medermycin, an analog of actinorhodin that lacks the C-8 hydroxyl group, to make mederrhodin A, which is hydroxylated at C-8 (36). Second, as shown here, ORF5 displays significant similarities with genes encoding flavin-dependent monooxygenases (Supplemental Fig. 1). Our results strongly support this hypothesis. With a pure preparation of ActVA, reported here for the first time, we clearly show that the ActVA-ActVB combination can catalyze an hydroxylation reaction using 1,5-dihydroanthraquinone as a substrate analog. Oxidation absolutely requires NADH, FMN, and molecular oxygen, supporting the notion that ActVB serves for the production of reduced flavins, whereas ActVA uses the latter to activate oxygen and oxidize the organic substrate. Accordingly, even though it was purified with no chromophore, we have shown that ActVA is able to bind FMN_ox, which displays a specific light absorption spectrum with a low energy transition at 438 nm. ActVA is also able to bind FMN_red, to which it provides protection from oxidation by air. With the true substrate, dihydrokalafungin, when it is available this in vitro system will allow us to conclude whether dimerization and hydroxylation is catalyzed by the same enzyme and, if not, which step occurs first. An interesting issue concerns the transfer of reduced flavin from one protein (ActVB) to another (ActVA), its control, and whether specific interactions between ActVB and ActVA are required for that purpose.

The K_d values for FMN both in the reduced and the oxidized form, characterizing their interaction with each component, ActVA and ActVB, have been carefully determined. This is the first time that all these values are given with this class of enzymes. They indicate that the flavin transfer between ActVA and ActVB is under a thermodynamic control, with a preference of FMN_ox for ActVB and a much stronger affinity of ActVA for FMN_red (K_d = 0.39 µM). The data showed that FMN_ox can be transferred from ActVA to ActVB (Fig. 6) and also that FMN_red generated in ActVB by reduction of FMN_ox either by NADH or by photoreduction can be removed from ActVB and transferred to ActVA. This was also in good agreement with the observation that the oxidation of ActVB·FMN_red by oxygen is greatly slowed down upon the addition of increasing amounts of apo-ActVA as a consequence of FMN_red transfer to ActVA where oxidation occurs slowly (Fig. 8).

All these results can, thus, be fitted into a very simple mechanism shown in Scheme 3. At the very beginning of the reaction, FMN_ox binds to ActVB, where it is reduced by NADH. The resulting FMN_red then diffuses out of ActVB and tightly binds to ActVA, where it reacts with oxygen. In this step a reactive intermediate is formed and used to hydroxylate the substrate, thus generating FMN_ox as will be discussed below. In the absence of the substrate it is likely that the intermediate decomposes to form hydrogen peroxide. Then in all cases FMN_ox diffuses out of ActVA to ActVB ready to start a new cycle. In this mechanism, based on a thermodynamic control of the reaction, we do not find it necessary to invoke a specific interaction between the two proteins and a complex channeling mechanism allowing FMN to travel from one protein to another without equilibrating with FMN free in solution. In fact we did not get any evidence for a complex between ActVB and ActVA. It is actually the case for most two-component flavin-dependent monooxygenase systems that the flavin reductase component and the monooxygenase component do not form a complex. Furthermore, in most cases the oxidoreductase component can be replaced by many flavin reductases, including the nonhomologous ones from other microorganisms. Therefore, direct protein-protein interactions are in general not essential.

View larger version (11K): [in this window] [in a new window]   SCHEME 3

	FOOTNOTES

 
^* 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1–4.

To whom correspondence may be addressed. Tel.: 33-4-38-78-91-03; Fax: 33-4-38-78-91-24; E-mail: mfontecave{at}cea.fr. To whom correspondence may be addressed. Tel.: 33-4-38-78-91-09; Fax: 33-4-38-78-91-24; E-mail: vniviere{at}cea.fr.

¹ The abbreviations used are: NTA, nitrilotriacetic acid; ORF, open reading frame; DHAQ, 1,5-dihydroanthraquinone.

² In the presence of both ActVB and ActVA, because FMN is in binding equilibrium with both proteins, the concentration of bound FMN can be calculated with the equation [FMN]_bound = ([FMN]_tot [ActVB]_free)/[(K_dActVB (1 + ([ActVA]_free)/(K_dActVA +)) [ActVB]_free)].

	ACKNOWLEDGMENTS

 
We acknowledge Yves Dupont, Vincent Forge, and Elizabeth Mintz for fruitful discussions. We thank Bernard Sartor for technical assistance. We thank Domenica Gräfin Von Der Schulenburg for reading the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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