Natural low-molecular mass organic compounds with oxidase activity as organocatalysts

Significance

Organocatalysts are low-molecular mass organic compounds composed of nonmetallic elements. Here, we report that actinorhodin (ACT), a bacterial-derived antimicrobial compound, acts as an organocatalyst, as indicated by the following findings: (i) substrate oxidation reactions that produced H₂O₂ proceeded in the presence of ACT; (ii) ACT was not consumed during the reactions; and (iii) a catalytic amount of ACT consumed an excess amount of the substrates. We propose that ACT kills bacteria by producing toxic amounts of H₂O₂. We also screened various ACT-like natural products and identified several that exhibited catalytic activity, suggesting that living organisms produce and use them as biocatalysts in nature.

Abstract

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of l-ascorbic acid and l-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H₂O₂ proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H₂O₂. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature.

A catalyst is a substance that increases the rate of a chemical reaction but is not consumed in the reaction. Catalysts are classified as homogeneous or heterogeneous, and most of the major homogeneous catalysts are metal-containing catalysts. In general, enzymes and ribozymes, which are classified as homogeneous catalysts, are referred to as biocatalysts. Enzymes are large proteins that catalyze numerous reactions in living organisms whereas ribozymes are composed of RNA and are known to catalyze phosphoryl transfer reactions (involved in RNA self-splicing), the formation of peptide bonds, and various other reactions (1–5). Organocatalysts, which are homogeneous catalysts, are low-molecular mass organic compounds derived from nonmetallic elements (e.g., carbon, oxygen, hydrogen, and nitrogen) (6). Organocatalysts are used in industry because they have the following advantages over metal-containing catalysts (7–10): (i) Organocatalysts exhibit catalytic activity under mild conditions (atmospheric pressure, room temperature, and neutral pH); (ii) disposal of depleted organocatalysts is inexpensive; (iii) environmental loads due to waste reaction mixtures containing organocatalysts are low; and (iv) the risk of product contamination by metal ions is low. Therefore, organocatalysts have been receiving much attention in the field of green chemistry (11).

In our laboratory, we have been studying the metabolism of actinomycetes from both fundamental and applied points of view (12–15). From Streptomyces, we discovered a previously unidentified enzyme, l-glutamate oxidase (EC 1.4.3.11) (16), which catalyzes the oxidation of l-glutamate to α-ketoglutarate. We are particularly interested in the isolation of molecules that catalyze oxidation reactions. Oxidation reactions are essential for living organisms and are important for industrial applications as well: For example, the first step of tryptophan metabolism is oxidation of tryptophan to kynurenine (17); oxidation reactions catalyzed by cytochrome P450 enzymes are involved in drug detoxification (18); and the oxidation of glucose by glucose oxidase is the first reaction that occurs in glucose sensors for measuring glucose concentrations in the blood. While screening for oxidases from streptomycetes, we discovered that actinorhodin (ACT) is a natural organocatalyst for oxidation reactions.

Results

Oxidation Activity of ACT.

Using a screening procedure involving a Clark-type oxygen electrode, with which O₂ concentrations can be monitored directly, we discovered that culture supernatants of Streptomyces coelicolor A3(2) catalyzed the oxidation of l-ascorbic acid (l-ASC) and l-cysteine (l-Cys) (Fig. S1 A and B, respectively). We initially attributed the activity to an unidentified oxidase, but we were unable to purify an enzyme from the supernatants. Interestingly, we observed a correlation between the depth of the color of the supernatants and their oxidase activity. Therefore, we turned our attention to ACT (Fig. 1A), a low-molecular mass organic compound that is the most well-known pigment produced by S. coelicolor A3(2) (19–23). We purified a large amount of ACT (Fig. S2) and investigated its oxidase activity. Surprisingly, we found that ACT catalyzed the oxidation of l-ASC and l-Cys (Fig. 1 B and C).

Fig. 1.

Catalytic activity of ACT for oxidation reactions. (A) Structure of ACT, which contains quinone moieties and has a molecular mass of 634. (B and C) Oxygen electrode analyses of reactions of ACT in the presence of 10 mM l-ASC (B) or 30 mM l-Cys (C) as the substrate. The reaction was initiated (black arrow) by the addition of 0.03 mM (final concentration) ACT (blue and red) or 1,4-dioxane (yellow). Catalase (140 units) was added (white arrow) during the reaction under the red-line conditions. (D) Oxygen electrode profile of ACT activity under the modified conditions with 30 mM l-ASC as the substrate. The reaction was initiated (black arrow) by the addition of 39.5 μM (final concentration) ACT. O₂ was bubbled forcibly into the reaction mixture whenever the O₂ concentration dropped below 0.06 mM.

To determine whether the catalytic activity was due to the presence of a metal, we quantitatively analyzed the metals in the ACT solution and 1,4-dioxane (which is used as the solvent for ACT in the activity assay) by means of inductively coupled radiofrequency plasma spectrometry. However, none of the 54 metals included in the assay (SI Materials and Methods) were detected, within the limits of the assay.

Next, we quantitatively analyzed ACT before and after the oxidation reaction by means of liquid chromatography–tandem mass spectrometry (Fig. S3). After a 20-min reaction with l-ASC as the substrate, the ionic strength (that is, the quantity) of ACT was 138, which was essentially the same as that before the reaction (136). During the same 20-min reaction, O₂ (235 nmol) was consumed completely whereas none of the ACT (39.5 nmol) was consumed. When 39.5 nmol of ACT was added to a reaction mixture containing 30 μmol of l-ASC as the substrate, the O₂ concentration decreased, and when O₂ gas was repeatedly bubbled into the reaction mixture over the course of 1 h, a total of 4.58 μmol of O₂, which was more than 100 times the amount of ACT, was consumed (Fig. 1D).

Identification of Reaction Products and Stoichiometry of Oxidation Reactions.

In the presence of l-ASC or l-Cys, ACT catalyzed oxidation reactions that consumed O₂ and produced H₂O₂. The expected products of these reactions were l-dehydroascorbic acid (l-DHA) and l-cystine (l-CSSC), respectively, and we used HPLCy (HPLC) to confirm the identity of the products. When l-ASC was the substrate, two new peaks derived from possible reaction products were observed, and their retention times were identical to those of authentic samples of l-DHA and H₂O₂ (Fig. S4 A and B, respectively). In contrast, when l-Cys was the substrate, a new peak with a retention time identical to that of an authentic sample of l-CSSC was observed (Fig. S4C), but H₂O₂ formation was not detected.

Using HPLC, we examined the stoichiometries of the oxidation reactions of l-ASC (2 mM) and l-Cys (2 mM), and the stoichiometry of O₂ consumption was monitored with an oxygen electrode. When l-ASC was the substrate, l-ASC and O₂ were consumed and l-DHA and H₂O₂ were formed in a 1:1:1:1 stoichiometry (Fig. 2A). When l-Cys was the substrate in the presence of ACT, l-Cys and O₂ were consumed and l-CSSC was formed in a 4:1:2 stoichiometry (Fig. 2B). The reaction of H₂O₂ with l-Cys reportedly has the following stoichiometry: 2l-Cys + H₂O₂ → l-CSSC + 2H₂O (24). Examination of the stoichiometry of l-Cys and H₂O₂ consumption and l-CSSC formation in the absence of ACT confirmed that l-Cys and H₂O₂ were consumed and l-CSSC was formed in a 2:1:1 stoichiometry (Fig. 2C). Combining the stoichiometries of the reactions shown in Fig. 2 B and C suggested that l-Cys and O₂ were consumed to form l-CSSC and H₂O₂ in a 2:1:1:1 stoichiometry. Although H₂O₂ reacted with l-Cys, it did not react with l-ASC.

Fig. 2.

Analysis of oxidation reaction stoichiometry. (A) Time courses of l-ASC oxidation and product generation in the presence of ACT: l-ASC (♢), O₂ (△), l-DHA (□), and H₂O₂ (×). (B) Time courses of l-Cys oxidation and product generation in the presence of ACT: l-Cys (♦), O₂ (△), and l-CSSC (○). (C) Time course of chemical reaction between l-Cys and H₂O₂ in the absence of ACT: l-Cys (♦), H₂O₂ (×), and l-CSSC (○). The methods used to analyze the substrates and products are described in Materials and Methods.

Effects of pH and Temperature on the Activity of ACT.

The pH dependence of the catalytic activity of ACT for the oxidation reactions was determined with l-ASC and l-Cys as substrates in various buffers. When l-Cys was the substrate, ACT exhibited maximum activity at pH 8.5 (Fig. 3A). When l-ASC was the substrate, ACT also exhibited the highest activity at pH 8.5, and l-ASC was oxidized spontaneously at pH values higher than 9.5 (Fig. 3B).

Fig. 3.

Effects of pH and temperature on the specific activity of ACT. (A and B) Oxidation reactions were carried out with l-Cys (A) or l-ASC (B) as the substrate at 30 °C in the following buffers (100 mM): glycine/HCl (□), citrate/NaOH (×), KPB (△), Tris⋅HCl (○), glycine/NaOH (♦), NaHCO₃/NaOH (■), and NaH₂PO₄/NaOH (●). (C) An ACT solution was preincubated at various temperatures for 1 h, and then its residual activity (♦) was assayed with an oxygen electrode. The dotted line indicates the catalytic activity of ACT without preincubation. (D and E) Oxidation reactions carried out at various temperatures with l-ASC (D) or l-Cys (E) as the substrate.

Next, we investigated the stability of ACT to heat treatment. An ACT solution was incubated at various temperatures for 1 h, and then the catalytic activity of the heat-treated ACT for oxidation reactions was assayed under the standard conditions. Even after heat treatment at 100 °C, the residual activity of ACT was 100% of the original activity, indicating that ACT was very stable (Fig. 3C).

We also evaluated the effect of oxidation reaction temperature on ACT activity. As the temperature of the oxidation reaction was increased, the concentration of dissolved O₂ in the reaction mixture decreased under the assay conditions used. To measure the catalytic activity of ACT for oxidation reactions at various temperatures, we modified the assay conditions so that O₂ gas was supplied to the reaction mixture forcibly. The specific activity of ACT was then calculated in terms of O₂ consumption, which was derived from the level of l-ASC consumption or l-CSSC formation (Fig. 3 D and E). We found that the catalytic activity of ACT increased exponentially in response to increasing reaction temperature. However, at temperatures over 90 °C, the reaction mixture started to boil so the activity could not be measured.

When we evaluated the catalytic activity of ACT for oxidation reactions at various l-Cys concentrations, we found that the activity increased linearly with increasing substrate concentration (Fig. S5).

Substrate Specificity.

The substrate specificity of ACT was assayed in a reaction mixture (1 mL) consisting of 100 mM potassium phosphate buffer (KPB, pH 7.4), 30 mM substrate, and 7.9 μM ACT. Among the tested substrates, l-ASC was the most suitable. Compared with the oxidation rate for l-ASC (100%), the rates for d-isoascorbic acid (86.2%), l-cysteine methyl ester (16.0%), DTT (9.8%), cysteamine (2.0%), thiosalicylic acid (1.1%), l-Cys (0.7%), dl-penicillamine (0.3%), and glutathione (0.2%) were all lower. Antioxidants (melatonin, rutin, protocatechuic acid, ferulic acid, vitamin A, d-α-tocopherol, sesamin, dibutylhydroxytoluene, curcumin, uric acid, coumarin, catechin, and chlorogenic acid) and various thiol compounds (glutathione disulfide, N-acetyl-l-cysteine, dl-homocysteine, thiomalic acid, and 2-mercaptobenzimidazole) were inert as substrates for ACT.

Inhibition of Bacterial Growth by ACT.

Some l-amino acid oxidases that produce H₂O₂ during oxidation reactions are known to show antibacterial activity (25, 26). Although ACT has antibiotic activity (27), its mechanism of action in bacteria remains unknown. In this study, we demonstrated that ACT also produces H₂O₂ in culture media. To determine whether the H₂O₂ produced by ACT showed antibiotic activity or not, we investigated the growth rates of Staphylococcus aureus NBRC 100910 (Biological Resource Center, NITE 100910) under seven sets of conditions (SI Materials and Methods). Because we expected H₂O₂ to be produced by ACT in the culture medium, catalase, which can eliminate H₂O₂, was added to several of the culture media. The bacteria grew normally when only KPB (instead of ACT, 1,4-dioxane, or catalase) was added to the culture medium (condition 7) (Fig. 4A). When 1,4-dioxane was added to the culture medium (condition 3), bacterial growth was slightly inhibited. Bacterial growth was lowest under condition 1, where ACT and 1,4-dioxane were added to the medium. The NBRC 100910 strain is known to be sensitive to ACT, and our findings confirmed that ACT did indeed show antibacterial activity. However, when catalase was added to the medium in the presence of ACT (condition 2), bacterial growth was restored somewhat compared with that observed under condition 1. In contrast, the addition of catalase to the medium containing 1,4-dioxane (condition 4) had no effect on the inhibition of bacterial growth by 1,4-dioxane. When 0.52 M H₂O₂ was added to the culture medium (condition 5), bacterial growth was inhibited for 12 h, but, by 24 h, bacterial growth was the same as that observed under condition 7. The addition of catalase to the culture medium containing H₂O₂ (condition 6) completely eliminated the effect of H₂O₂ on bacterial growth.

Fig. 4.

Growth curves for S. aureus NBRC 100910 cultured under various conditions. (A) Condition 1 (blue), ACT and 100 mM KPB (pH 7.4) (instead of catalase); condition 2 (red), ACT and 70 units of catalase; condition 3 (green), 1,4-dioxane (solvent for ACT) and 100 mM KPB (pH 7.4); condition 4 (purple), 1,4-dioxane and 70 units of catalase; condition 5 (gray), 0.52 M (final concentration) H₂O₂ and 100 mM KPB (pH 7.4); condition 6 (orange), 0.52 M (final concentration) H₂O₂ and 70 units of catalase; and condition 7 (yellow), none of 100 mM KPB (pH 7.4). Growth was measured by determining the average optical density at 600 nm (OD₆₀₀) for three independent cultures of each strain at each time point (the range and average of each group of measurements for each strain are shown). (B) Condition a (blue), ACT and 100 mM KPB (pH 7.4) (instead of catalase); condition b (green), 1,4-dioxane (solvent for ACT) and 100 mM KPB (pH 7.4); condition c (purple), 1,4-dioxane, 100 mM KPB (pH 7.4), and H₂O₂ (each 0.01 mM fed); condition d (orange), 1,4-dioxane, 100 mM KPB (pH 7.4), and H₂O₂ (each 0.25 mM fed); condition e (red), 1,4-dioxane, 100 mM KPB (pH 7.4), and H₂O₂ (each 0.3 mM fed); condition f (yellow), 1,4-dioxane, 100 mM KPB (pH 7.4), and H₂O₂ (each 0.32 mM fed); and condition g (gray), 1,4-dioxane, 100 mM KPB (pH 7.4), and H₂O₂ (each 0.36 mM fed). Growth was measured by determining the average optical density at 600 nm (OD₆₀₀) for three independent cultures of each strain at each time point (the range and average of each group of measurements for each strain are shown).

Next, to estimate the amount of H₂O₂ produced by ACT in the culture medium, we investigated the bacterial growth rates in media containing 1,4-dioxane and H₂O₂ at various concentrations (Fig. 4B) (SI Materials and Methods). During cultivation, aliquots of H₂O₂ were added to the medium every 2 h. S. aureus NBRC 100910 grew normally when H₂O₂ was added periodically at low concentration (0.01 mM), but the bacteria did not grow when the concentration of added H₂O₂ was higher than 0.36 mM. Bacterial growth in the presence of ACT was inhibited compared with that when 0.25 mM H₂O₂ was added periodically. This finding suggests that 1 mmol of ACT produced about 120 mmol of H₂O₂ during cultivation in these media.

Screening of Other Natural Products for Catalytic Activity.

Using an oxygen electrode, we screened a selection of other natural products from bacteria, plants, and animals to determine whether they had any catalytic activity for oxidation reactions. Each compound was dissolved in dimethyl sulfoxide at a concentration of 10 mM for the assay. The consumption of O₂ in reaction mixtures containing 30 mM l-ASC or l-Cys, 100 mM KPB (pH 7.4), and 10 μM natural product was measured by the method used to determine ACT activity. Three plant-derived natural products, 2,6-dimethoxyquinone, antiarol, and juglone, were found to consume more than 50 μM O₂ (corresponding to more than five times the amount of the compound consumed in the reaction), demonstrating that these compounds had catalytic activity (Fig. 5).

Fig. 5.

Organocatalysts derived from living organisms. Structures of natural organocatalysts and their catalytic activities.

Discussion

While screening streptomycete cultures for new oxidases using a Clark-type oxygen electrode, we found that ACT, which is a low-molecular mass blue pigment produced by S. coelicolor A3(2), catalyzed the oxidation of both l-ASC and l-Cys. Because metal complexes have been reported to show oxidase-like catalytic activity (28), we performed metal analysis on the purified ACT, which was found to contain no metals. We determined that the amount of ACT after the reaction was the same as that before the reaction and that the amount of O₂ consumed in the presence of ACT and excess substrate was more than 100 times the amount of substrate consumed in 1 h, verifying the turnover of ACT. These findings indicated that ACT is an organocatalyst.

The products of the ACT-catalyzed oxidation reactions were identified as follows. When l-ASC was the substrate, ACT catalyzed the following reaction: l-ASC + O₂ → l-DHA + H₂O₂ (Fig. 2A). Previously, l-ASC was reported to be the only substrate of “synthetic” quinones as organocatalysts. In addition, a possible mechanism, referred to as the “non-enzyme quinone redox cycle,” for the reaction has been proposed (29, 30). Considering the fact that ACT contains a quinone moiety, we suggest that ACT-catalyzed oxidation occurs by the same mechanism. When l-Cys was the substrate of ACT, the following reaction occurred: 4l-Cys + O₂ → 2l-CSSC + 2H₂O (Fig. 2B). H₂O₂, which was expected to be one of the products, was not detected by means of HPLC. When catalase (which produces O₂ from H₂O₂) was added to the reaction mixture, the O₂ consumption was reduced by half, suggesting the presence of H₂O₂ in the reaction mixture. Indeed, the following reaction proceeded under these conditions: 2l-Cys + H₂O₂ → l-CSSC + 2H₂O (Fig. 2C). These findings indicate that, during the oxidation reaction, H₂O₂ was produced and then rapidly eliminated by means of spontaneous reaction with l-Cys. In other words, ACT catalyzed the following oxidation reaction: 2l-Cys + O₂ → l-CSSC + H₂O₂.

We also investigated the properties of ACT from enzymological standpoints. ACT exhibited maximum activity at pH 8.5, and the oxidation reaction proceeded at room temperature under atmospheric pressure. In addition, ACT retained its original activity even after being heated at 100 °C for 60 min. When O₂ was forcibly bubbled into the reaction medium, ACT activity increased exponentially with increasing reaction temperature. These findings show that the catalytic activity of ACT for oxidation reactions increased in a temperature-dependent manner. Moreover, because the specific activity of ACT increased linearly with increasing substrate concentration, the maximum rate (V_max) for ACT could not be calculated from double-reciprocal plots of reaction rate versus substrate concentration. These characteristics strongly suggest that ACT behaves as an organocatalyst. ACT exhibited substrate specificity in oxidation reactions: the specific activity of ACT when l-ASC was the substrate differed from that when l-Cys was the substrate. In addition, when we examined the ability of ACT to catalyze the oxidation of various thiol compounds and antioxidants, we found that thiol compounds, like l-Cys, could be used as substrates.

There have been many reports on the development of functional organocatalysts (31, 32). Among such catalysts, l-proline is the only known natural organocatalyst; it has been shown to catalyze asymmetric aldol reactions (33–35), but only in organic solvents or boiling water (36), conditions that are impossible in living organisms. Therefore, although l-proline is present in nature, it probably cannot catalyze such reactions in vivo. To the best of our knowledge, our discovery that ACT, a natural product of microbial origin, acts as a biocatalyst under physiological conditions is the first such discovery to be reported.

ACT is a benzoisochromanequinone antibiotic (19). Many studies of its biosynthesis have been reported (20–23); however, there have been only a few reports on its antibacterial activity (27), and its mechanism of action remains unknown. Some l-amino acid oxidases, which catalyze oxidation reactions in which H₂O₂ is among the products, have been reported to show antibacterial activity (25, 26). H₂O₂ formation is considered to be the cause of this activity. Synthetic quinones, which undergo a nonenzymatic redox cycle that produces H₂O₂, show cytotoxicity toward living cells (37). Because we found that ACT also produces H₂O₂, we assumed that H₂O₂ formation was the source of the antibiotic activity of ACT. To confirm this possibility, we examined the effect of catalase (which can remove H₂O₂) on bacteria treated with ACT (Fig. 4). When S. aureus NBRC 100910 was exposed to ACT, its growth was inhibited, but when the bacteria were exposed to ACT in the presence of catalase, growth was slightly higher than that in the absence of catalase. These findings indicate that growth inhibition by ACT (i.e., its antibiotic activity) in the absence of catalase was due to the toxicity of H₂O₂ (produced by the oxidation of unidentified organic chemicals present in the bacteria or the supernatant). When S. aureus NBRC 100910 was grown in the presence of ACT and catalase, the catalase decreased the level of H₂O₂ and rescued the bacterial growth. The growth rate under these conditions did not reach that in the presence of catalase and absence of ACT, indicating that ACT produced H₂O₂ constantly on the bacterial surface or within the cells. The continuous production rate of H₂O₂ in the broth was estimated to be nearly 0.15 μmol/h. Although antibiotics target many different bacterial functions and growth processes, to the best of our knowledge, the mechanism of action of all known antibiotics involves inhibition of the biosynthesis of substances necessary for life, such as nucleic acids (DNA and RNA), proteins, and components of the bacterial cell wall (38, 39). In contrast, the mechanism of action of ACT involves the production of H₂O₂, which causes bacterial death, and this unprecedented mechanism differs from the mechanisms of known antibiotics.

To date, there have been no reports of the use of natural products produced by living organisms as organocatalysts. Interestingly, the pH at which ACT activity was optimal (pH 8.5) was the same as the pH of the supernatant of S. coelicolor A3(2) after culture for 5 d. This finding suggests that this strain uses ACT as an organocatalyst. Furthermore, we speculate that other organisms may also use organocatalysts such as ACT, for the following reasons: (i) ACT catalyzes oxidation reactions, which are generally important in vivo (e.g., metabolism of amino acids, lipids, nucleic acids, and drugs); (ii) l-ASC and l-Cys, which are substrates of ACT, are common compounds in living organisms; and (iii) ACT has a quinone structure, which plays an important physiological role in vivo (40, 41). Therefore, we screened various natural products derived from bacteria, plants, and animals with the goal of finding additional new organocatalysts. Three compounds showed over five turnovers per molecule; none of these compounds contain a metal, and they all have a quinone skeleton, suggesting that they may function as organocatalysts in nature. Our findings open the door on a new field of organocatalysts present in living organisms.

Organocatalysts including l-proline have been studied for industrial uses (10, 42). Organocatalysts have some advantages over enzymes and metal catalysts: They show high-temperature stability, and they work under mild conditions (at room temperature, in aqueous solutions, and at neutral pH). Under similarly mild conditions, ACT catalyzed the oxidation of l-ASC and l-Cys and produced H₂O₂. H₂O₂, a versatile oxidizing agent, is an important commodity chemical. On an industrial scale, H₂O₂ is synthesized by means of an anthraquinone autooxidation process. However, this process requires metal catalysts, high temperature, and high pressure to reduce the oxide-anthraquinone. Therefore, improvements of the process are desirable from a green-chemistry viewpoint (43). We suggest that ACT could be used in a new process for producing large amounts of H₂O₂ from the oxidation of l-ASC (or l-Cys) under mild conditions.

Materials and Methods

Detailed descriptions of the materials, bacterial strains and media, metal analysis, quantitative analysis of ACT during the reaction by liquid chromatography–tandem mass spectrometry, effects of pH and temperature on the activity of ACT, bacterial growth in culture media containing ACT, bacterial growth in culture media containing different concentrations of H₂O₂, and screening of organocatalysts from natural products are given in SI Materials and Methods.

Assaying of Catalytic Activity of ACT for Oxidation Reactions.

Measurement of O₂ consumption with an oxygen electrode.

The catalytic activity of ACT for oxidation reactions was measured with an oxygen electrode (Hansatech Instruments Ltd.), which monitors the O₂ concentration. The reaction mixture was composed of 2 mM, 10 mM, or 30 mM l-ASC or l-Cys, 100 mM potassium phosphate buffer (KPB) (pH 7.4), and 10 μL ACT solution, in a final volume of 1 mL The reaction was initiated by injecting the ACT solution into an electrode cuvette and was carried out at 30 °C. Because slight spontaneous oxidation of substrates occurs, the decrease in the O₂ concentration was always measured as a negative control when 1,4-dioxane (solvent for ACT) was used in the reactions instead of the ACT solution. One unit of ACT is defined as the amount of the compound that catalyzes the consumption of 1 μmol of O₂ per min.

Measurement of substrate consumption and product formation by HPLC.

For the oxidation reaction of l-ASC, the reaction mixture comprised 2 mM l-ASC, 100 mM KPB (pH 7.4), and 7.9 μM ACT, in a final volume of 5 mL The reaction was started by the addition of ACT and was carried out at 30 °C. The reaction was stopped by the addition of an equal volume of 0.2 M HCl. l-ASC and the product [l-dehydroascorbic acid (l-DHA)] were analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a ZIC-HILIC column (4.6 i.d. × 150 mm; Merck KGaA) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent (100 mM ammonium acetate: CH₃CN = 3:7); flow rate, 1.0 mL/min; and photodiode array detector, 240 nm. Hydrogen peroxide (H₂O₂) was analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a Shoudex SUGER KS-801 column (8.0 i.d. × 300 mm; Showa Denko K.K.) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent (2 mM Na₂SO₄, 20 nM EDTA); flow rate, 0.75 mL/min; and electrochemical detection (ECD700S; Eicom Corp.).

For the oxidation reaction of l-Cys, 2 mM l-Cys was used instead of 2 mM l-ASC. l-Cys and the product [l-cystine (l-CSSC)] were analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a CAPCELLPAK column (4.6 i.d. × 150 mm; Shiseido Company, Ltd.) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent [3.5 mM sodium 1-heptanesulfate, 0.1% (wt/vol) phosphate: CH₃CN = 95:5]; flow rate, 1.0 mL/min; and photodiode array detector, 210 nm.

Isolation and Purification of ACT.

S. coelicolor A3(2) was inoculated for the first subculture. The first subculture was carried out at 28 °C for 1 wk with reciprocal shaking in a 500-mL shaking flask containing 100 mL of YEME media. Then, 10 mL of the first subculture was inoculated into a 2-L shaking flask containing 500 mL of YEME media. The second culture was also performed at 28 °C with reciprocal shaking. After incubation for 1 wk, the cells were harvested by centrifugation at 10,400 × g at 4 °C and washed with 500 mL of 1 M HCl, and then stirred in 800 mL of 1 M NaOH. The solubilized blue pigment was separated from the mycelial debris by centrifugation. The supernatant was acidified to pH 2∼3 with concentrated (conc.) HCl, which yielded a red precipitate of crude ACT. The precipitate was collected by centrifugation and washed with acetone, followed by filtration. The collected pigment was dried in vacuo and recrystallized from 1,4-dioxane. The resultant ACT was dissolved again in 1,4-dioxane, and then the ACT solution was analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a TSK-gel ODS-100S column (4.6 i.d. × 150 mm; Tosoh Corporation) under the following conditions: column temperature, 40 °C; flow rate, 1.0 mL/min; photodiode array detector, 190∼600 nm; mobile-phase solvent A [0.5% (vol/vol) acetic acid in CH₃CN] and solvent B [0.5% (vol/vol) acetic acid in deionized H₂O], and subsequent gradient elution. The mobile-phase composition was 65% solvent A/35% solvent B at 0 min, 65% solvent A/35% solvent B at 5 min, 5% solvent A/95% solvent B at 30 min, 5% solvent A/95% solvent B at 35 min, and 35% solvent A/65% solvent B at 40 min. Determination of the molecular mass of the purified ACT was performed by LC-ESI-MS with a Shimadzu LCMS-8030 system equipped with a TSK-gel ODS-100S column under the above condition. The MS analysis data were acquired with a mass spectrometer with ESI in the negative modes.

Stoichiometry of the Chemical Reactions Between Substrates and H₂O₂.

For the chemical reaction between l-ASC and hydrogen peroxide (H₂O₂), the reaction mixture comprised 0.5 mM l-ASC, 0.25 mM H₂O₂, and 100 mM KPB (pH 7.4), in a final volume of 5 mL. The reaction was started by the addition of H₂O₂ and carried out at 30 °C. The reaction was stopped by the addition of an equal volume of 0.2 M HCl. l-ASC was analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a ZIC-HILIC column (4.6 i.d. × 150 mm) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent (100 mM ammonium acetate: CH₃CN = 3:7); flow rate, 1.0 mL/min; and photodiode array detector, 240 nm. H₂O₂ was analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a Shoudex SUGER KS-801 column (8.0 i.d. × 300 mm) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent (2 mM Na₂SO₄, 20 nM EDTA); flow rate, 0.75 mL/min; and electrochemical detection (ECD700S).

For the reaction between l-Cys and H₂O₂, 0.5 mM l-Cys was used instead of 0.5 mM l-ASC. l-Cys was analyzed by HPLC with a Shimadzu LC-10Avp system equipped with a CAPCELLPAK column (4.6 i.d. × 150 mm) under the following conditions: column temperature, 40 °C; isocratic elution; mobile-phase solvent [3.5 mM sodium 1-heptanesulfate, 0.1% (wt/vol) phosphate: CH₃CN = 95:5]; flow rate, 1.0 mL/min; and photodiode array detector, 210 nm.