Studies on Kojic Acid Metabolism by Microorganisms

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1 [Agr. Biol. Chem., Vol. 35, No. 13, p , 1971] Studies on Kojic Acid Metabolism by Microorganisms Part XI. Comenic Aldehyde Dehydrogenaset õ (5-Methoxy Comenic Aldehyde Dehydrogenase) By Jun IMOSE, Seiichi NONOMURA and Chuji TATSUMI Agricultural Technology, Department of Agriculture, University of Osaka Prefecture, Sakai, Osaka,,Japan Received Tune 7, 1971 An enzyme, comenic aldehyde dehydrogenase, which catalyzes the oxidation of comenic aldehyde to comenic acid was partially purified from cell extract of Arthrobacter ureafaciens K-1. The enzyme was purified 31-fold at Sephadex G-100 filtration step, 112-fold at DEAE- Sephadex A-50 fractionation step, and recovery of the activity was 73.3% and 38.5%, respectively. NADP and magnesium ion were essential for the oxidation. The enzyme shows optimum activity at ph 7.8. Enzyme activity was extremely sensitive to sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. L-Cysteine or dithiothreitol protected the enzyme from p-chtoromercuribenzoate inhibition. Carbonyl reagents, such as hydroxylamine and semicarbazide, inhibit the enzyme reaction by formation of addition compounds between carbonyl reagents and aldehyde group of the substrate. The enzyme was completely inactivated after heating for 5 min at 40 C. The Km for 5-methoxy comenic aldehyde is 2.5 x 10-6 M, and for NADP is 0.4 ~10-6M. The reaction product, 5-methoxy comenic acid was identified by paperchromatography. The characterization of the enzyme has been carried out by using 5-methoxy comenic aldehyde as the substrate in stead of comenic aldehvde. In the course of our studies on kojic acid degradation by Arthrobacter ureafaciens K-1 utilizing kojic acid as a sole carbon source, some of the intermediates have been found.' As the results of degradation of kojic acid by resting cells, we proposed a degradation pathway as follows; Kojic acid Comenic alde hyde Comenic acid_??_4d-galacturonic acid Tagaturonic acid_??_ Of this pathway, we already reported partial purification and some properties of D-galacturonate ketol-iso merase2) and kojic acid oxidase.3) This paper deals with partial purification and some properties of comenic aldehyde de hydrogenase, which forms comenic acid by oxidation of the aldehyde when K-1 is grown with kojic acid. In order to avoid the difficulty of preparation of comenic alde hyde, 5-methoxy comenic aldehyde was used throughout the experiments. t Studies on ƒá-pyrone Compounds (Kojic Acid) Metabolism by Microorganisms. (Part XIV) 1) S. Nonomura and C. Tatsumi, Nippon Nogeika gaku Kaishi, 35, 954 (1961); idem, ibid., 38, 213 (1964); idem, ibid., 38, 216 (1964). 2) J. Imose, S. Nonomura and C. Tatsumi, Nippon Nogeikagaku Kaishi, 41, 94 (1967). 3) S. Nonomura, J. Imose and C. Tatsumi, Agr. Biol. Chem., 33, 1223 (1969); J. Imose, S. Nonomura and C. Tatsumi, ibid., 34, 1443 (1970).

2 2026 J. IMOSE, S. NONOMURA and C. TATSUMI MATERIALS AND METHODS Microorganism. Arthrobacter ureafaciens K-1 main tained on a bouillon agar slant containing 0.2% kojic acid was used. Cultural conditions. In shaking culture, K-1 was grown in a 500 ml volume flask containing 100 ml of a bouillon medium consisting of kojic acid, 0.2%; meat extracts, 0.500; peptone, 0.1%; NaCl; 0.5%; adjusted to ph 7.2 with KOH and also a synthetic medium consisting of kojic acid, 0.2%; KH2PO4, 0.2%; NH4NO3, 0.100; MgSO4.7H2O, ; yeast extracts, o; adjusted to ph 7.2 with KOH. In static culture, K-1 was grown in a 500 ml volume flask containing 200 ml of the media described above. After sterili zation, I ml inoculum of overnight culture was added and then incubated either at 30 Ž for 24 hr with shaking or at 30 C for 70 hr without shaking. Enzyme assay. The enzyme reaction was followed by measuring the rate of formation of NADPH at 340 mis at room temperature with a Shimazu spectro photometer model QV-50 and with a Nippon Denshi Unicorder model U-100. Unless otherwise specified, the standard incubation mixture contained the following constituents in the final volume of 3 ml: phos phate buffer, ph 7.8, 100ƒÊmoles; MgSO4 E7H2O, 0.3ƒÊmole; NADP, 0.2ƒÊmole; an appropriate amount of enzyme; and 5-methoxy comenic aldehyde, 1.5 ƒê moles. The reaction was started at room temper ature by addition of the aldehyde into the reaction mixture which contained all other components except the aldehyde and preincubated. Dilution of the enzyme was always done by using 50 mm phosphate buffer, ph 7.8. Protein estimation. The amount of protein was spectrophotometrically estimated by the method of Warburg and Christian. Materials. 5-Methoxy comenic aldehyde,5) 5- methoxy comenic acid6) and N-tris(hydroxymethyl) methylglycine(tricine)7) were synthesized as described in the references, respectively. RESULTS (I) Induction of comenic aldehyde dehydrogenase Specific activity of comenic aldehyde de hydrogenase (Sephadex G-100 fraction) pre pared from K-1 grown in various culture media and under various culture conditions, are shown in Table I. The highest specific activity is obtained in kojic-synthetic medium with shaking. This condition was chosen for the preparation of the enzyme. (II) Purification of comenic aldehyde dehydrogenase The results of the preparation of comenic aldehyde dehydrogenase are summarized in Table II. The procedures of all steps were performed at 0--5 C. Step 1. Preparation of cell extract Cells were harvested by centrifugation at 5,000 x g for 5 min, washed twice with sterilized TABLE I. EFFECT OF COMPOSITION OF MEDIA AND CULTURE CONDITIONS ON COMENIC ALDEHYDE DEHYDROGENASE ACTIVITY The enzyme activity was determined in a standard reaction mixture under the standard assay condition. Sephadex G-100 fraction was used as the enzyme solution. 4) O. Warburg and W. Christian, Biochem. Z., 310, 384 (1941). 5) R. Kotani and C. Tatsumi, Bull. Univ. Osaka Pref. Ser B., 10, 33 (1960). 6) I. Ichimoto, T. Washino, F. Fujii and C. Ta tsumi, Nippon Nogeikagaku Kaishi, 41, 317 (1967). 7) N. Good, Arch. Biochem. Biophys., 96, 653 (1962).

3 Studies on Kojic. Acid Metabolism by Microorganisms. Part XI 2027 TABLE II. PURIFICATION OF COMENIC ALDEHYDE DEHYDROGENASE a ) One unit of the enzyme activity is defined as the amount catalyzing the formation of mƒêmole of NADPH per min. water, suspended in 0.05 M phosphate buffer, ph 7.8, and stored in frozen state. The thawed cells were disrupted by sonication for 15 min, followed by centrifugation at 12,000 ~g for 10 min. After the cell debris was removed, cell extract was obtained. Step 2. Protamine sulfate treatment Cell extract was adjusted to ph 6.0 with acetic acid. Freshly prepared 2% protamine sulfate solution (1 mg of protamine sulfate per 10 mg of enzyme protein) was added to cell extracts with stirring. Stirring was continued for 20 min and the precipitates were removed by centrifugation at 12,000 x g for 10 min. The supernatant obtained was adjusted to ph 7.8 with 1 N KOH, and used as a protamine sulfate fraction. Step 4. Sephadex G-100 filtration A column (1.8 ~45.0cm) was packed with Sephadex G-100 equibrated with 0.05 M phos phate buffer, ph 7.8. The enzyme preparation obtained in Step 3 was applied to the column and eluted with the above buffer at a flow rate of 0.3 ml per min. Fractions with the highest specific activity were collected. The elution profile of protein concentration and, comenic aldehyde dehydrogenase activity are shown in Fie. 1. Step 3. Ammonium sulfate fractionation The resulting supernatant solution was brought to 50% saturation by gradual addition of solid ammonium sulfate, and after stirring for 15 min, the suspension was centrifuged at 12,000 x g for 10 min, and the precipitates were discarded. Then solid ammonium sulfate was further added to 60% saturation. The result ing precipitates having enzyme activity were collected by centrifugation at 12,000 ~g for 10 min and dissolved in 0.05 M phosphate buffer, ph 7.8. The enzyme solution obtained was used as an ammonium sulfate fraction. FIG. 1. Sephadex G-100 Gel Filtration of Ammoni um Sulfate Fraction. œ \ œ Absorbance at 280 mp œ--- œ Comenic aldehyde dehydrogenase activity.

4 2028 J. IMOSE, S. NONOMURA and C. TATSUMI FIG. 2. Chromatography of Sephadex G-100 Gel Filtration Fraction on a DEAE- Sephadex A-50 Column. œ \ œ Absorbance at 280mƒÊ. œ--- œ Comenic aldehyde dehydrogenase activity. - E- E- KCl concn. Step 5. DEAE-Sephadex A-50 fractionation A column (1.8 ~40.0cm) was packed with DEAE-Sephadex A-50 equilibrated with 0.05 M phosphate buffer, ph 7.8. The enzyme pre paration obtained in Step 4 was applied to the column and the 20 ml of the same buffer was added into the column to wash the enzyme. A gradient was applied with 100 ml of the buffer in mixing flask and 500 ml of the buffer TABLE III. EFFECT OF METAL ION ON COMENIC ALDEHYDE DEHYDROGENASE ACTIVITY Each metal ion was added to the standard reaction mixture, and the reaction mixture was incubated at room temperature for 3 min. Sephadex G-100 fraction was used as the enzyme solution. The enzyme activity was determined under the standard assay condition. containing 0.5 M KCl in a reservoir. Fractions with the highest enzyme activity were collected at a rate of 0.4 ml per min. The elution pro file of protein concentration and comenic alde hyde dehydrogenase activity are shown in Fig. 2. The specific activity was increased to 31-fold in Step 4 (45.2-fold in the highest activity, fraction No. 11), and 112-fold in Step 5 (121- fold in the highest activity, fraction No. 33), and the recoveries of the activity were 73.5% and 38.5%. (III) Properties of comenic aldehyde dehydrogenase (a) Requirements for enzyme activity. The enzyme is specific for NADP, and NAD could not serve as a hydrogen acceptor in place of NADP. It should be noted (Table III) that Mg" ion activates the enzyme and to a lesser extent, Mn2+ ion.

5 Studies on Kojic Acid Metabolism by Microorganisms. Part XI 2029 FIG. 3. Comenic Aldehyde Dehydrogenase Activity as a Function of Enzyme Concentration. Straight line shows comenic aldehyde dehydro genase activity calculated by method of least minimum square. FIG. 5. Effect of Temperature on Comenic Aldehyde Dehydrogenase Activity. The enzyme solution (Sephadex G-100 fraction) was treated at various temperature for 5 min, and after cooling, the enzyme activity was determined using a standard reaction mixture. (c) Optimum ph. The comenic aldehyde dehydrogenase activity has optimum at ph 7.8 as shown in Fig. 4. This value was in close agreement for four buffers except Tris. The relative rates at given ph values were slower in borax, veronal and tricine than in phosphate. However, the velocity of comenic aldehyde oxidation increased rapidly with increase in ph (up to 9.0) without attaining a maximum in Tris buffer. FIG. 4. Effect of ph on Comenic Aldehyde De hydrogenase Activity. The enzyme solution (Sephadex G-100 fraction) was incubated at various ph values in a standard reaction mixture. œ \ œ Potassium phosphate buffer. œ--- œ Borax-KH2PO4 buffer. \ Tris-HCl buffer. \ Veronal buffer. --- Tricine buffer. (b) Rate of NADP reduction as a function of enzyme concentration. As shown in Fig. 3, the rate of reduction of NADP was directly pro portional to the enzyme concentration. (d) Heat stability. The enzyme solution was heated for 5 min at each temperature in dicated, and cooled immediately after treat ment. The enzyme activity remaining was determined. Figure 5 indicates a relatively low resistance of the enzyme to heating. The activity decreased rapidly as the temperature increased and loss of 87%? of the activity was found upon heating 40 Ž for 5 min. (e) Effect of inhibitors. The enzyme is in. hibited by reagents reacting with sulfhydry groups, such as p-chloromercuribenzoate anc monoiodoacetate, and in particular, the enzyme is sensitive to p-chloromercuribenzoate (Table IV). It may, therefore, be granted that the

6 2030 J. IMOSE, S. NONOMURA and C. TATSUMI TABLE IV. EFFECT OF INHIBITORS dithiothreitol or L-cysteine. This result strongly suggests that sulfhydryl groups were close to or in the active site of the enzyme. Carbonyl reagents, such as hydroxylamine, semicarbazide and sodium bisulfite, reacted with carbonyl groups of the substrate, and inhibited the aldehyde oxidation. (f) Effect of magnesium ion. As shown in Table III, Mg2+ or Mn2+ ion activates the enzyme. In Fig. 6, the reduction rate of NADP increased up to 1 mm of Mg2+ ion. TABLE V. PROTECTIVE EFFECT OF L-CYSTEINE OR DITHIOTHREITOL (DTT) AGAINST PCMB INHIBITION Experiment I indicates that the enzyme solution was preincubated with PCMB, and then the enzyme reaction was started by addition of substrate. Experiment 2, 3, 4 and 5 indicate that the enzyme solution was preincubated with L-cysteine, and after PCMB addition, the enzyme reaction was started by addition of substrate. In each experiment, Sephadex G-100 fraction was used as the enzyme solution. FIG. 6. Effect of Magnesium Ion on Comenic Aldehyde Dehydrogenase. Reaction mixture was incubated at room temper ature for 3 min. Sephadex G-100 fraction was used as the enzyme solution. The activity was determined under the standard assay condition. ctive site of the enzyme bears sulfhydryl roups. As shown in Table V, enzyme inactivation _??_y PCMB was protected in the presence of (g) Substrate specificity. The substrate spe cificity of this enzyme is narrow as shown in Table VI. It was also examined whether this enzyme would oxidize aromatic aldehyde, such as salicyl-aldehyde or benzaldehyde, but the results were negative for all of aldehydes tested. (h) Michaelis constants. Michaelis constant was found to be 2.5 ~10-6M for 5-methoxy

7 Studies on Kojic Acid Metabolism by Microorganisms. Part XI 2031 TABLE VI. SUBSTRATE SPECIFICITY a) Crude crystals were used as the sample. FIG. 8. Determination of Velocity Constant with Respect to NADP. FIG. 7. Determination of Velocity Constant with Respect to 5-Methoxy Comenic Aldehyde. comenic aldehyde, and 0.4 ~10-6M for NADP (Figs. 7 and 8). (IV) Identification of reaction product 5-Methoxy comenic acid was identified as the product by paperchromatography of the ether extracts of incubation mixture. The incubation mixture contained 150ƒÊmoles of 5-methoxy comenic aldehyde, enzyme solution, 20ƒÊmoles of NADP, 30ƒÊmoles of MgSO4 7H2O and 2.0 mmoles of phosphate buffer, ph 7.8 in the final volume of 60 ml, and incu bation was carried out at room temperature FIG. 9. Paperchromatogram of the Reaction Product. A; Sample (ether extracts) B; 5-Methoxy comenic aldehyde C; 5-Methoxy comenic acid Solvent; n-butanol : acetic acid : water (5:2:3 v/v) for 30 min. The incubation mixture was de proteinzed with 1 ml of sulfuric acid (sulfuric acid-water, 1 : 3, v/v), and the products were extracted with ether for 24 hr. The extracts were dissolved in a small volume of ethanol,

8 2032 J. IMOSE, S. NONOMURA and C. TATSUMI and then subjected to paperchromatography with solvent system: n-butanol-acetic acid-water (5:2:3v/v). The compounds were detected by spraying bromphenol blue reagent or 2,4- dinitrophenyl hydrazine. By spraying the latter, a spot was detected at a Rf value of 0.86, and was identified with authentic 5- methoxy comenic aldehyde. This was the unaltered substrate (Fig. 9). DISCUSSION Aldehyde dehydrogenase which oxidizes aldehydes to their respective acids has gener ally required NAD or NADP as coenzyme. Aldehyde dehydrogenase from yeast8) and benzaldehyde dehydiogenase from Pseudomonas fluorescens9) were NADP-specific. Comenic aldehyde dehydrogenase from Arthrobacter ure afaciens K-1 described in this report was also active only with NADP. Aldehyde dehydrogenase from Pseudomonas aeruginosa10) requires Fe2+ or Ca2+ ion and that from yeast8) requires Ca2+, Mg2+, Ba2+ or Mn2+ ion. It is generally recognized that aldehyde dehydrogenase is dependent on K+ ion for its activity. Comenic aldehyde dehydrogenase from Arthrobacter ureafaciens K-1 does not require K+ ion, but requires Mg2+ or Mn2+ ion. From the point of view that this enzyme requires NADP and Mg2+ ion, it may be different from bacterial aldehyde dehydro genase, and may be similar to yeast aldehyde dehydrogenase reported by Seegmiller.8) Aldehyde dehydrogenase from Pseudomonas aeruginosa10,11) oxidizes various aliphatic and aromatic aldehydes, and benzaldehyde dehydro genase from Pseudomonas fluorescens9) oxidizes benzaldehyde and salicyl aldehyde, but not 8) J. E. Seegmiller, J. Biol. Chem., 201, 629 (1953). 9) C. Stachow, I. Stevenson and D. Day, ibid., 242, 5294 (1967). 10) M. T. Heydeman and E. Azouulay, Biochim. Biophys. Acta, 77, 545 (1963). 11) R. Von Tigerstrom and W. Razzel, J. Biol. Chem., 243, 2691 (1968). acetaldehyde. However, comenic aldehyde dehydrogenase can not oxidize benzaldehyde and salicyl aldehyde, and oxidize slightly ascetaldehyde. This enzyme may have absolute specificity for aldehyde adjacent to ƒá-pyrone ring. It is reported by Monder12) that the velocity of enzymatic methylglyoxal oxidation increased rapidly with ph without attaining a maxi mum in Tris, since Tris may form an addition compound with carbonyl group of the sub strate. In comenic aldehyde dehydrogenase, increase of absorbance at 340mƒÊ may also indicate the formation of an addition compound between Tris and carbonyl group of comenic aldehyde. Tricine buffer may not form such addition compound and a low activity was observed. It is reported by Jackoby13) and Tigerstrom11) that aldehyde dehydrogenase is inhibited by arsenite, which reacts with sulfhydryl groups. But inhibition of comenic aldehyde dehydro genase by arsenite could not be demonstrated. It is known that sodium cyanide not only reacts with carbonyl group to form cyanhydrin, but also acts as chelating agent and degrades the substrate in case of methylglyoxal.12) In comeic aldehyde dehydrogenase, low concent ration of potassium cyanide inhibited the enzyme completely. It is not apparent whether cyanide forms cyanhydrin, serves as chelating agent, or reacts with sulfhydryl group of the enzyme. Comenic aldehyde dehydrogenase isolated from Arthrobacter ureafaciens K-1 may be dif ferent from aldehyde dehydrogenase from bacteria by the specificity of requirement for metal ion, and has been found to be similar to yeast aldehyde dehydrogenase prepared by Seegmiller8) with respect to requirement of NADP and Mg2+ ion. But substrate specificity of this enzyme is narrow, and the enzyme may act only to aldehyde adjacent to ƒá-pyrone ring. 12) C. Monder, J. Biol. Chem., 242, 4603 (1967). 13) W. Jackoby, ibid., 232, 89 (1958).

colorimetrically by the methylene blue method according to Fogo and manometrically. In the presence of excess sulfur the amount of oxygen taken up

colorimetrically by the methylene blue method according to Fogo and manometrically. In the presence of excess sulfur the amount of oxygen taken up GLUTA THIONE AND SULFUR OXIDATION BY THIOBACILLUS THIOOXIDANS* BY ISAMU SUZUKI AND C. H. WERKMAN DEPARTMENT OF BACTERIOLOGY, IOWA STATE COLLEGE Communicated December 15, 1958 The ability of Thiobacillus