Amino Acid Metabolism in Microorganisms Part IV L

[Agr. Biol. Chem., Vol. 32, No. 6, p. 727-733, 1968]

Amino Acid Metabolism in Microorganisms

Part IV L-Methionine Decarboxylase Produced by a Streptomyces Strain

By Hiroshi HAGINOand Kiyoshi NAKAYAMA

Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd.

Received December 1, 1967

3-Methylthiopropylamine (MTPA) formation from L-methionine in Streptomyces sp. K37

was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of L-methionine. The properties of the enzyme were studied in detail using acetone dried

cells or cell-free extract. The enzyme was specific for L-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The

optimum pH for the reaction was 6.0-8.0 and the optimum temperature was about 40°C.

Carbonyl reagents (10-2-..10-3 M) inhibited the reaction completely, and silver nitrate and mercuric chloride (10-3•`10-4M) markedly inhibited the reaction. Km value for the

reaction was 1.21 X 10-5 M. L-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.

As previously reported", certain Streptomyces using acetone dried cells or cell free extract strains isolated from soil accumulated 3- of Streptomyces sp. K37. L-Methionine assay

methylthiopropylamine (MTPA), decarboxy using this decarboxylase was also attempted. lated product of methionine, when they were This paper deals with the results of these cultured in the medium containing L- or DL- experiments. methionine. Now, MTPA formation from L-methionine by Streptomyces sp. K37 was MATERIALS AND METHODS further studied in detail. MTPA formation Strains. An unidentified strain, Streptomyces sp. was confirmed to be the reaction catalyzed by K37 isolated from soil was used.

L-methionine decarboxylase (L-methionine Culture method. Thirty millilitre of a seed carboxylyase). As far as authors know, medium in a 250 ml Erlenmyer flask was inoculated enzymic decarboxylation of L-methionine has with the spores from a Bennett-agar slant and never been reported though oxidative decar incubated at 30°C for 48 hr. The seed medium boxylation of L-methionine was studied in contained 2% glucose, 1%, yeast extract, 1% cabbage"" and horse raddish4,5) by M. peptone, and 0.25 0 sodium chloride (pH 7.4 before sterilization). The seed culture was transferred into Mazelis. Therefore, the properties of the a 250 ml Erlenmeyer flask containing 20 ml of a decarboxylase of L-methionine was examined growth medium and the flask was incubated at 30°C on a rotary shaker for 96 hr. The growth medium 1) H. Hagino and K. Nakayama, Agr. Biol. Chem., had the following composition: glucose 1%, K2HPO4 31, 1369 (1967). 0.05 00, MgSO4•E7H2O 0.025%, (NH4)2SO4 0.2% 2) M. Mazelis, Biochem. Biophys. Res. Commum., 1, 59 (1959). yeast extract 0.3 0„ CaCO3 0.3 0,,' and L-methionine 3) M. Mazelis, Federation Proc., 19, 4 (1960). 5 mg/ml (pH 7.4 before sterilization). 4) M. Mazelis, J. Biol. Chem., 237, 104 (1962). Preparation of acetone dried cells or cell free 5) M. Mazelis and L. L. Ingraham, ibid., 237, 109 (1962). extract. The cells grown in the growth medium 728 Hiroshi HAGINO and Kiyoshi NAKAYAMA

for 6-8 hr were harvested and washed twice with TABLE I. STOICHIOMETRY OF DECARBOXYLATION

saline water, and then acetone dried cells were OF L-METHIONINE

prepared by treating the washed cells with cold acetone. The washed cells were suspended in 1/15 M

phosphate buffer (pH 6.0) to be the cell concentration 10---15 mg (dry weight) per millilitre. The cells were disrupted by sonic treatment for 20 min at 0°C

with a Ultra Sonic Oscillator (Kaijo Denki Co.). Cellular debris was removed by centrifugation at

15,000 X g for 30 min, and the clear supernatant

fraction was obtained. Then, ammonium sulfate Reaction mixture: acetone dried cells (60mg/ml) 1 ml, 1/15M phosphate buffer (pH 6.0) 1.3ml, was added to the supernatant in a concentration of L-methionine (3-5 mg/ml) 0.5 ml, (4.5 N HCl 0.2 50% saturated. The preciptate obtained was ml) dissolved into 0.01 M phosphate buffer and dialyzed Reaction was carried out at 37.5°C for 10 min. against the same buffer for 18 hr at 5•Ž. The dia lyzed fraction was used for the enzyme assay. from L-methionine accompanied equimolecular Enzyme assay. Enzyme activity was determined CO, liberation. When the decarboxylation manometrically by measuring CO2 liberation by a reaction was carried out in the reaction Warburg manometer or spectrophotometrically by deter- mining MTPA produced. In the manometeric method, mixture containing 0.2 ml of 20 % KOH in

the main compartment contained the enzyme solution the centre well, CO, liberated was absorbed

(0.5---1.0 ml)with appropriate amount of buffer (1.3- into KOH and little gas evolution was 1.5 ml) and pyridoxal phosphate solution (0.3ml), a observed. These facts suggest that the side-arm contained L-methionine solution (0.5 ml) and reaction is simple decarboxylation of L- the other side-arm contained 4.5 N HC1 (0.2 ml). The methionine other than the oxidative decar total volume of the reaction mixture was 3.0 ml, boxylation reported by M. Mazelis2•`5) The reaction was cerried out at 37.5"C. To liberate 2. Decarboxylase activity and residual L-me the retained CO2, 4.5 N HCL (0.2 ml) was tipped into thionine. the reaction mixture at the end of the experiment. As previously reported", MTPA forming In the assay of L-methionine by the decarboxylase method, acid tip was not used and the CO2 retained activity was induced in the existence of L- was corrected by calculation. In the photometric methionine in the growth medium and was

method, MTPA formed was measured by ninhydrin found in the cells incubated for short period reaction according to the modified method of Giris) (6-8 hr) but little activity was found in the after isolating the MTPA from residual L-methionine cells incubated for longer period. Therefore, by paperchromatography using the solvent system; the decarboxylase activity in the cell was n-butanol: acetic acid: water (4: 1:2). A minute examined in relation to the concentration of amount of MTPA was determined by the color residual L-methionine in the broth. As shown development with picric acid according to the in Fig. 1, the decarboxylase activity in the Richter's method7) after extracting the MTPA from cell decreased in parallel with the rapid con the reaction mixture at alkaline pH with ether. sumption of L-methionine in the medium. RESULT New supplement of L-methionine to the broth 1. Stoichiometry of MTPA formation from L- after 6, 12, and 24 hours' incubation retained methionine. or increased the decarboxylase activity of the As shown in Table I, MTPA formation cell.

3. Substrate specificity. 6) K. V. Girl, A. N. Radharishnan and C. S. V L-Methionine decarboxylase in Streptomyces ardyanathan, Anal. Chem., 24, 1677 (1952). 7) D. Richter, M. H. Lee and D. Hill, Biochem. J sp. K37 was tested for the substrate specificity ., 35, 1225 (1941). with acetone dried cells. As shown in Table Amino Acid Metabolism in Microorganisms. Part IV 729

Therefore, the enzyme was confirmed to be the decarboxylase specific to L-methionine . 4. Optimum pH Fig. 2 shows the velocities of decarboxyla tion with acetone dried cells in different reaction pH. The optimum pH was 6--8 .

FIG. 1. Variation of the Enzyme Activity with Age of Culture.

: Enzyme activity of the cells supplemented L-methionine (5 mg/ml) to the culture broth (initial L-methionine conc. was 5mg/ml) after 6, 12, and 24 hours' incubation. FIG. 2. Effect of pH on the Activity of L-Methio -•›-: Enzyme activity of the cells without nine Decarboxylase. new supplement of L-methionine.

TABLE II. SPECIFICITY OF THE L-METHIONINE DECARBOXYLASE Reaction mixture: acetone dried cell (45 mg/ml) 1ml, buffer solution 1.3ml, L-methionine (6 mg/ml) 0.5ml, (4.5N HCl 0.2ml). Reaction was carried out for 10 min at 37.5°C.

Reaction mixture: acetone dried cells (60mg/ml) 1.0ml, 1/15M phosphate buffer (pH 6.0) 1.3ml, amino acid solution (5mg/ml) 0.5ml, (4.5 N-HCl 0.2ml) FIG. 3. Effect of Temperature on the Activity of Reaction was carried out at 37.5°C for 10 min. L-Methionine Decarboxylase.

Reaction mixture: enzyme solution 0.5 nil, 1/15M II, only L-methionine among 22 amino acids phosphate buffer (pH 6.0) 1.7ml, L-methionine tested was decarboxylated and the small (6mg/ml) 0.5 ml, pyridoxal phosphate (50ƒÊg/ml) 0.3ml. quantity of gas evoluted from some other amino Reaction was carried out at different temperature acids may be due to experimental error. 10 min.

---•›--- 730 Hiroshi HAGINO and Kiyoshi NAKAYAMA

Subsequent experiments were carried out at TABLE IV. EFFECT OF ENZYME INHIBITORS (I) pH 6.0 (1/15M phosphate buffer). 5. Optimum temperature. Fig. 3 shows the variation of the velocity of decarboxylation with temperature. The optimum temperature for the reaction was approximately 40°C. 6. Stimulation of the decarboxylation by pyridoxal phosphate. Participation of pyridoxal phosphate as a cofactor was reported in the known amino acid decarboxylation. Therefore, the effect of pyridoxal phosphate on the decarboxylation Reaction mixture: enzyme solution 0.5ml, 1/15M of L-methionine was tested using dialyzed phosphate buffer (pH 6.0) 1.65 ml, L-methionine enzyme preparation. As shown in Table III, (5mg/ml) 0.3ml, inhibitor solution 0.2ml, pyridoxal phosphate (50 fig/ml) 0.15ml (4.5 N HCl pyridoxal phosphate markedly stimulated the 0.2ml) decarboxylation. Reaction was carried out at 37.5°C for 10 min.

TABLE III. EFFECT OF PYRIDOXAL PHOSPHATE TABLE V. EFFECT OF ENZYME INHIBITORS (II) ON THE ACTIVITY OF L-METHIONINE DECARBOXYLASE

Reaction mixture: enzyme solution 0.5 ml, 1/15M phosphate buffer (pH 6.0) 1.7ml, L-methionine (5mg/ml) 0.3ml, pyridoxal phosphate solution 0.3ml (4.5 N HCl 0.2ml) Reaction was carried out at 37.5°C for 10 min.

7. Enzyme Inhibitors. Various enzyme inhibitors were tested for Reaction mixture: enzyme solution 0.5 ml, 1/15M the effect on the decarboxylation of L- phosphate buffer (pH 6.0) 1.65ml, L-methionine (5mg/ml) 0.3ml, metalic salts solution 0.2ml, methionine. As shown in Table IV, carbonyl pyridoxal phosphate (50ƒÊg/ml) 0.15ml, (4.5 N HCl reagents such as hydrazine, hydroxylamine, 0.2ml) isonicotinic acid hydrazide, and semicarbazide Reaction was carried out at 37.5°C for 10 min.

(10-2•`10-3M) inhibited the decarboxylation completely, while p-chloromercuribenzoic acid (10-3•`10-4M) slightly inhibited the reaction.

(10-4M) was without effect. 8. Protective effect of pyridoxal phosphate on Table V shows the inhibitory effect of the enzyme against heat inactivation. heavy metalic salts. Mercuric chloride and Enzyme inactivation by heating for 30 min silver nitrate (10-3•`10-4M) heavily inhibited at various temperature was shown in Fig. 4. the decarboxylation of L-methionine. Cupric Below 30°C, the enzyme was stable, but was sulfate, ferrous sulfate, and manganese chloride partially inactivated above 40°C. Then, Amino Acid Metabolism in Microorganisms. Part IV 731

Fig. 5, pyridoxal phosphate was effective, while D-methionine was not. 9. Km value. From the Lineweaver-Burk plots shown in Fig. 6, the Michaelis constant for the crude enzyme was estimated to be 1.21 x 10-'m.

FIG. 4. Effect of Heat on the Stability of L- Methionine Decarboxylase.

Reaction mixture: 1/15 M phosphate buffer 1.5ml, enzyme solution 0.5ml, L-methionine (6mg/ ml) 0.5ml, pyridoxal phosphate (50ƒÊg/ml) 0.3ml, (4.5 N HCl 0.2ml). Reaction was carried out at 37.5°C with enzyme. FIG. 6. Lineweaver-Burk Plot for L-Methionine Solution kept for 30 min in the water bath at Decarboxylase. each temperature.

10. L-Methionine assay using the decarboxylase. Application of the decarboxylase described above to L-methionine assay was attempted. Time course of CO, liberation with some concentrations of L-methionine was shown in Fig. 7. With acetone dried cells (15 mg/ml),

FIG. 5. Protective Effect of Pyridoxal Phosphate against Enzyme Inactivation by Heat.

After enzyme solution containing pyridoxal phosphate or D-methioninc was kept in the water bath at 50°C for 3060 min, enzyme activity was FIG. 7. Time Course of CO2 Evolution with Varia assayed. tion of L-Methionine Concentration. Reaction mixture: acetone ciriea cells too mg/ml) pyridoxal phosphate and D-methionine were 1 ml, 1/15M phosphate buffer 1.5 ml, L-methio tested for their protective effect against heat nine solution 0.5 ml. Reaction was carried out at 37°C. inactivation of the enzyme. As shown in 732 Hiroshi HAGINO and Kiyoshi NAKAYAMA

TABLE VI. RECOVERY TEST was applied to the solutions of yeast extract OF L-METHIONINE ASSAY and casamino acids. The determination was

performed using 0.5 ml of the sample con taining L-methionine at the concentration below 1000ƒÊg/ml. Table VII shows the com

parison of the results obtained by the de carboxylase method with those obtained by the sodium nitroprusside reagents" A good coincidence was noted between the both values obtained by the two methods with the Decarboxylation of L-methionine was carried out at yeast extract though 10 % difference was noted 37.5°C for 90 min in the reaction mixture having with the casamino acids. the following composition; acetone dried cells (45mg/ml) 1ml, 1/15M phosphate buffer (pH 6.0) 1.5ml, L-methionine solution 0.5 ml. DISCUSSION MTPA formation from L-methionine in

TABLE VII. EXAMPLES OF L-METHIONINE ASSAY Streptomyces sp. K37 was confirmed to be the WITH L-METHIONINE DECARBOXYLASE reaction catalyzed by L-methionine decarboxy lase (L-methionine carboxy-lyase) according to the fact that MTPA formation accompanied equimolecular CO, liberation. The enzymic decarboxylation of L-methionine is a new reaction and the physiological meaning of the role of the enzyme awaits to be elucidated. Amino acid decarboxylases studied have

Decarboxytion of L-methionine was carried out at been reported to require pyridoxal phosphate 37°C for 90 min in the reaction mixture having as a cofactor. In L-methionine decarboxylase, the following composition; acetone dried cells participation of pyridoxal phosphate was evi (45mg/ml) 1ml, 1/15M phosphate buffer (pH 6.0) 1.5ml, sample (diluted to 10•`20-fold) 0.5ml. denced indirectly by the following facts: i) Decarboxylation catalyzed by dialyzed enzyme was stimulated by pyridoxal phosphate ii) evolution of CO, was measured for 90 min. Pyridoxal phosphate protected the enzyme The amount of CO, evolved reached 97.5 % against heat inactivation iii) Carbonyl rea of the theoretical yield with 500ƒÊg of L-methio nine 91% with 1000ƒÊg, and 86% with 1500ƒÊg. gents which were known to be competitive inhibitors with pyridoxal phosphate against As the concentration of L-methionine in Schiff base formation heavily inhibited the creased, CO, yield decreased. For the ac decarboxylation. But using more purified curate determination of L-methionine, it is enzyme, the requirement of pyridoxal phos desirable to react L-methionine below the concentration of 500ƒÊg. Then, the recovery phate for the reaction must be followed directly. of L-methionine in different concentrations L-Methionine assay using the decarboxylase added to the samples was examined. As was attempted. The advantage of the enzymic shown in Table VI, L-methionine added at assay generally lies in its simpleness, its speed, the concentration below 250ƒÊg was recovered with an error less than 2 %, but the recovery of L-methionine added in a concentration of 8) T. E. McCarthy and M. X. Sullivan, J. Biol. 500ƒÊg was76.3 %. Then, L-methionine assay Chem., 141, 871 (1941). Amino Acid Metabolism in Microorganisms. Part IV 733 its stereospecificity, and the fact that it can tion below 500ƒÊg per flask owing to the be performed on crude sample without any weakness of the enzyme activity used, the treatment. Although the samples must have method described is a new pratical method been diluted to the L-methionine concentra for specific determination of L-methionine.