Some properties of p-coumarate decarboxylase from Cladosporiumphlei'
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Dt,ptrt.ttriettt r~f'Agric~rrltrrrrrl Biolo.qy, Frrc~rItyqfAgriclrlt~rr~, Hokkrrido Utti\~et..sity,S ~ r p ~ ) o rJoO , ~LIII AND
Y. MINO Lnhortrtoty of Ettl~irotttttet~tol Bottrtty, Doprrrtt71c~tttr ~ f A g r o e t ~ ~ ~ i r o t t n tSi.irttce. t ~ t t t r ~ l Obihiro Ut~iversity,Obilziro, Hokkrrirlo, Jtrprrtt Accepted May 7, 1976 HARADA,T . , and Y. M I N O . 1976. Some properties of p-coumar;~te dec;~rboxylase from Cltrdo.s~~orirrtnplilri. Can. J. Microbial. 22: 1258-1262. The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 1 3 "C. respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 "C f o r 5 minand by half at 25 "C in 24 h, but kept almost unchanged at -20 " C a t least for lodays. The activity was not inhibited by potassium cyanide. sodium diethyldithiocarbamate. ethylenediaminetetraacetic acid disodium salt, o r sodium citrate at 10 m M concentration, but was inhibited by p-chloromercuribenzoate o r iodoacetate at 0. I m M , the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid, cinnamic acid, o r p-methoxycinnamic acid, but not by fitmaric acid. acrylic acid, p-hydroxystyrene, furcatin, p-hydroxyphenylacetic acid, or phloretic acid. An ~~nsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. K , value for trrrtrs- p-coumaric acid was a b o ~ l 6.5 t x 10-4 M. HARADAT , . , et Y . M I N O . 1976. Some properties of p-coumarate decarboxylase from Clrrclo.sporirrtitpltlci. Can. J . Microbiol.22: 1258-1262. Le pH et la temperature optimales de la p-coumarate dkcarboxylase sont de 6.0 et 23 "C respectivement. L'activite de I'enzyme est rCduite aux trois quarts par un traitement thermique a 35 "C pendant 5 min et de rnoitie en 24 h a 25 "C; cependant, il n'y a presque pas de changement h -20 "C, pendant au moins I0 jours. L'activite n'est pas inhibee par le cyanure de potassium, le sodium diethyldithiocarrnate. le sel disodique de I'acide ethyltne-diamine-tetrahtique ou le citrate de sodium i~ 10 m M . Toutefois elle est inhibee par le p-chloromercuribenzoate ou I'iodoacetate B 0. I mM: I'inhibition par le p-chloromercuribenzoate est empcchee grandement par la presence du glutathion reduit ou le dithiothreitol. L'activite est inhibee par I'acide maleique, I'acide cinnamique ou I'acide 11-methoxycinnamique, mais ne I'est pas par I'acide fumarique, I'acide acrylique. lep-hydroxystyrene, la furcatine, I'acidep-hydroxyphCnylac6tique ou I'acide phloretique. Un groupement p-hydroxy non-substitue sur I'anneau benzene et une chaine laterale d'acide acrylique sont requis pour I'activite de I'enzyme. La valeur K , pour I'acide tt,trt~s-p-coumariqueest, approximativement. 6.5 x 10-WM. [Traduit par le journal]
Introduction Enzymes decarboxylating cinnamic acid derivatives have been demonstrated to occur in several microorganisms ( I , 3 , 4 , 7). In a previous paper (4), we reported that the timothy leaf spot fungus, Cladospori~inzphlei, has an ability to decarboxylateTPCA2 to p-hydroxystyrene. Some properties of p-coumarate decarboxylase from Aerobacter species have been reported (3), but little is known about the enzymes from fungal sources. This paper described some chemical and physical properties of p-coumarate decarboxylase from C. phlei. 'Received October 14, 1975. 2rrntrs-p-Coumaric acid.
Materials and Methods Flrtignl C~~ltrrre mtrl Enz~jtt~e Prc~pnrrrtiott The timothy leaf spot fungus Clar~osporirut~ plrlei de Vries was used. This fungus was isolated from a local variety of timothy (Pltler~tiipratetrsc, L.) in the Tokachi district in 1969 and identified by Dr. T . Narita (Obihiro University). Stock cultures were kept as slants on the potato agar at 25 " C in the dark for a month. The potato agar slant was prepared as follows. T w o hundred grams o f minced potato tubers were boiled in 1 litre of water for half an hour, followed by filtration through gauze. Twenty grams of glucose and 15 g of pulverized agar were added to the filtrate diluted t o 1 litre with water a n d a 7-ml portion in a test t ~ ~ was b e sterilized at 120°C for 20 min. From this stock culture, a loopful of the fungus was inoculated into a 50-ml erlenmeyer flask containing 20 ml of the liquid medium consisting of 10.0 g glucose, 2.0 g casaniino acid, 1.2 g Na,CO3.10 H 2 0 , 1.0 g KH2P0,, a n d 0.5 g MgS0,.7H20, per litre, a n d 0.5 m g
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HARADA A N D MINO
pyridoxine, 0.1 mg thiamine, 0 2 nig FeS0,.7H20, 0.2 mg ZnSO,.7H,O, and 0.2 mg MnS0, 7 H 2 0 , per litre, and cultured wlthout shaking at 25°C in the dark for 20 days. The liquid medlum was adjusted to pH 6 0 with I N HCl and then sterilized at 120 "C for 20 min before use. The cultured fungus was washed with deionized water. After removing excess water on the surface with filter papers, 20 g of the wet cells were ground thoroughly in 5 ml of 0.1% NaCl solution with 0.5 g of quartz sand in a mortar at 0 "C, followed by centrifugation at 25 000 g at 2 "C for 10 min. Two millllitres of the supernatant solution was chromatographed on a Sephadex G-100 column (1.5 cm x 30 cm) using 0.1 M NaCl solutlon as eluant at a flow rate of 30 ml/h. The eluate was collected In 5-mI fractions. Elutlon profiles of the enzyme and protein are shown in Fig. I. Fractions in tubes Nos. 23-39 were comblned and used as the enzyme solution (0.4 mg of protein/ml). Protein concentration was determined by the method of Lowry et a/. (6) with bovine serum albumln used for a standard.
TUBE N o
FIG. I . Elution profiles of protein (broken line) and enzyme activity (solid line) after Sephadex G-100 column chromatography. The enzyme activity was expressed as the evolution of C 0 2 / I ml of enzyme solution in each fraction per first 30 min.
Assny of the Etrzynle Actiurly The enzyme activlty was determined by measuring the evolution of carbon dloxide during the first 30 min at 20 OC in the reaction mixture consisting of I ml each of enzyme solution containing 0.4 mg of protein and phosphate buffer (67 mM, pH 6.0) in the main chamber and 0.3 ml of 50 niMTPCA (final concentration, 6.5 m M ) in the sidearm, unless otherwise stated. After the manometers were equilibrated for 15 min, the reactlon was initiated by the addition of a substrate from a sidearm. Each measurement was corrected with a proper blank.
Clletnicals D~hydrocaffeicand dihydroferulic acids were prepared from the correspond~ngunsaturated acids, respectively, by hydrogenation in 80% methanol solutlon in the presence of a palladium catalyst. p-Hydroxystyrene was synthesized by the method of Dale and Hennis (2) p-Methoxycinnamic acid was provlded by Mr. M. Terazawa (Obihiro Universlty) and furcatin by Dr. H. Iniazeki (Nagoya Universlty). The other chemicals obtained commercially were used without further ourlfica-
Results Ccr~,bon Dioxide Evolirtion from TPCA by the Enzytlze As shown in Fig. 2, decarboxylation from TPCA proceeded almost linearly during the first 30 min and thereafter decreased gradually until decarboxylation ceased completely after 2 h. pH and Tenzperature The enzyme activity above pH 6.5 was determined by measuring the evolution of total carbon dioxide after acidifying the reaction mixture with 0.2 ml of 1 N H,SO, using a manometer flask with two sidearms. The optimal pH and temperature of the enzyme were around 6.0 (Fig. 3) and 23 "C (Fig. 4), respectively.
30
60
90
120
MINUTE
FIG. 2. Evolution of carbon dioxide from TPCA.
FIG.3. Effect of pH on the enzyme activity. Open and closed circles indicate Mcllvaine (citrate-phosphate) and phosphate buffers, respectively. The enzyme activity was expressed as the evolution of C0,/0.4 mg of protein per first 30 min.
Eflect of Heat Treatment Two millilitres of the enzyme solution in a test tube was shaken (120 rpm) in a water bath at a given temperature for 5 min and then cooled
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CAN. J. MICROBIOL. VOL. 2 2 , 1976
TABLE2. Effect of storage temperature on the enzyme activity
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Rel. % act. Storage period, days
20 @C*
0 "C*
- 20 "C*
NOTE:T h e enzyme activity measured immediately after preparation was e x ~ r e s s e das loo'%.. 15
20
25
30
35
enzyme activity was tested using a Inanometer flask with two sidearms. As shown in Table 3, PCMB3 or iodoacetate at 0.1 mM concentration promptly with cold water. As shown in Table 1, inhibited the activity, but the inhibition by exposure of the enzyme solution to 60°C for PCMB was markedly protected in the presence 5 min brought about the complete disappearance of reduced glutathion or dithiothreitol a t 1 mM. of the activity and the activity was reduced to This suggests that SH groups in the enzyme about three quarters even at 35 "C for 5 min, molecule are required for activity. indicating that the enzyme was comparatively Inhibition by TPCA Analogs heat-labile. The enzyme activity was examined in the Changes in the Etlzyme Activity Stored at Varied presence of a compound related structurally to TPCA. The activity was inhibited by rnaleic, Te17~perat~ires cinnamic, orp-methoxycinnamic acid, but not by The enzyme solution containing each of 1 m M chloramphenicol and dihydrostreptomycin (as fumaric acid, acrylic acid, p-hydroxystyrene, antimicrobial agents) was stored at different furcatin, p-hydroxyphenylacetic acid, or phloretic temperatures for designed periods. As shown in acid. Table 2, the activity was -reduced by half at Substrate Specificify 20 "C and was slightly lost even at 0 "C in 24 h. Substrate specificity was examined using both a non-heat-treated and a heat-treated (35 "C for Effect of Metal Chelatorsand Sulfhydryl Inhibitors The enzyme activity was not inhibited by 5 min) enzyme preparation. The relative decarpotassium cyanide, sodium diethyldithiocarba- boxylation rates are presented in Table 5. mate, ethylenediaminetetraacetic acid disodium Among the compounds tested, cis-p-coumaric, salt, or sodium citrate at 10 m M concentration, bans-p-coumaric, caffeic, and ferulic acids were suggesting that metal ions are not involved in the decarboxylated. These results reflect the fact that activity. The effect of sulfhydryl inhibitors on the an unsubstituted 4-hydroxy group on the benzene ring and an acrylic acid side chain are required for enzyme activity. Decarboxylation TABLE1. Effect of heat-treatment rate was decreased by the introduction of a on the enzyme activity substituent into the 3-position of the benzene Treatment ring. Carbon dioxide evolution by heat-treated temp., "C Rel. % act. enzyme was nearly three quarters of that by nonheat-treated enzyme preparation irrespective of the substrate used, indicating that all compounds decarboxylated are probably attacked by the same enzyme. TEMPERATURE ('C)
FIG.4. Effect of temperature on the enzyme activity.
NOTE: T h e enzyme activity was measured manometrically at 20°C as previously described. T h e activity after a 5-min incubation a t 30°C (88 p1 C02) was expressed as 100%.
K,,, Value The Michaelis constant for TPCA was calcu3p-Chloromercuribenzoate.
HARADA AND MINO
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TABLE3. Effect of SH inhibitors on the enzyme activity SH inhibitor
Rel. % act.
None (TPCA only) Iodoacetate (0.1 m M ) PCMB (0.1 mM) PCMB (0.1 mM) + reduced glutathione (I mM) PCMB (0.1 mM) + dithiothreitol (I mM)
100 32 0 68 78
NOTE:The activity in the absence of any SH inhibitor was expressed as 100%. Immediately after adding a SH compound, PCMB and TPCA were simultaneously added to the enzyme solution. The final concentration of TPCA was 6.5 m M in each case. The concentrations of SH inhibitors and SH compounds were also final ones.
TABLE4. Effect of TPCA analogs Analogs, 5 rnM
Rel. % act.
None (TPCA only) Maleic acid Fumaric acid Acrylic acid p-Hydroxystyrene Furcatin Cinnamic acid p-Methoxycinnarnic acid Phloretic acid
100 82 100 100 100 100 56 72 100
SUBSTRATE I
NOTE:The enzyme acuvlty In the absence of any TPCA analog was expressed as 100Z The solution contalnlng TPCA and an analog, whlch was prevlously adjusted to pH 6.0, was added to the enzyme solut~on. The final concentration of TPCA was 6 5 m M In each case
CONCENTRATION (.10'~)
FIG. 5. K,,,value for TPCA. The value was calculated from a Lineweaver-Burk plot.
lated to be about 6.5 x lop4 M from a Lineweaver-Burk plot as presented in Fig. 5.
Discussion Some chemical and physical properties of pcoumarate decarboxylase from C. phlei were
examined. TPCA decarboxylation by the enzyme proceeded almost linearly during the first 30 min and thereafter decreased gradually (Fig. 2). The enzyme is probably considered to be inactivated in the course of the reaction, since the product p-hydroxystyrene did not inhibit the activity. Though the stability of p-coumarate decarboxylase from other sources has not been reported,
TABLE5. Substrate specificity COz evolut~on
Substrate a c ~ d Cinnamic / I nn~-p-Couniarlc cis-p-Coumaric 111-Coumar~c o-Coumaric Phloretic p-Methoxycinnarnic Caffeic Dihydrocaffeic Ferulic Dihydroferullc Sinapic Acrylic Crotonic
Non-treated (N)
Treated at 35 "C (T)
0 90 140 0 0 0 0 50 0 40 0 0 0 0
0 70 95 0 0 0 0 35 0 30 0 0 0 0
T/N ratio* 0.77 0 68
0.70 0.75
NOTE Figures gl\en are C 0 2(vl) evolved during the first 30 mln F ~ n asubstrate l concentrailon was 6 5 mM. * C 0 2 evolution by heat-treated enzyme/C02 e\olut~onby non-heat-treatcd enzyme.
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CAN. J. MlCROBlIOL. VOL. 22. 1976
the C , phlei enzyme seems to be fairly labile, since the enzyme was slightly inactivated even by heat treatment at 35 "C (Table I) and reduced by half at 25 "C in 24 h after enzyme preparation (Table 2). Furthermore, treatment by ammonium sulfate and chromatography on diethylaminoethyl (DEAE)-cellulose or Dowex 50 brought about its complete inactivation and considerable activity was lost during dialysis against tap water (data are not detailed here). This lability may be explained in terms of the oxidation of sulfhydryl groups required for the activity (Table 3). Cladospori~in~ plilei decarboxylase was inhibited by maleic acid, but not by fumaric acid as was the case for Aerobacter enzyme (3). Whiting and Carr (7) reported the bacterial decarboxylation of TPCA through a reductive route involving a hydroxyphenylpropionic acid as an intermediate. In contrast with their results, the decarboxylation of TPCA by C. phlei proceeds without any modification of the double bond in the acrylic side chain (4). The formation of styrene from cinnamic acid by some molds (1, 5) has been reported. The enzyme from C. phlei is considered to be different from those enzymes, judging from the results presented in Table 5. Aerobacter decarboxylase ( 3 ) was reported to require a relatively unhindered 4hydroxy group on the aromatic ring and an acrylic side chain. A similar situation appears to
be the case for the C , plzlei enzyme. In this study, enzyme purification has been unsuccessful because of instability. The problems mentioned above remain to be clarified by further purified enzyme.
Acknowledgments We are indebted to Prof. Y. Nishijima for reading this manuscript. We also thank Miss Y. Nabara for her technical assistance.
I. CLIFFORD, D. R., J. K. F A U L K E R J., R. L. WALKER, and D. WOODCOCK. 1969. Metabolism of cinnamic acid by Aspergilllrs niger. Phytochemistry. 8: 549-552. 2. DALE,W. J., and H. E. HENNIS.1958. Substituted styrenes, 111. The syntheses and somechemical properties of vinylphenols. J. Am. Chem. Soc. 80: 3645-3649. , J., J. C. L E W I SJ. , W. CORSE,and R . E. 3. F I N K L EB. L U N D I N1962. . Enzyme reactions with phenolic compounds: formation of hydroxystyrenes thro~lghthe decarboxylation of 4-hyd~.oxycinnamic acids by Aerobtrcrer. J. Biol. Chem. 237: 2926-293 1. 4. HARADA.T., and Y. MINO. 1973. Formation of 4-hydroxystyrene frornp-coumaricacid by the timothy leaf spot fungus, Cltrtlospor-ilrm phlei. Ann. Phytopathol. Soc. Japan, 39: 438-440. 5. JAMINET,F. 1950. Hydrocarbonic fermentation in a tolu sirup mixture. J. Pharm. Belg. 5: 191-201. 0 . H.. N . J. ROSEBROUGH, A . L. FARR,and R. 6. LOWRY, J. RANDALL. 1951. Protein measurementwith the Foiin phenol reagent. J. Biol. Chem. 193: 265-275. 7. WHITING, G. C., and J. G. CARR.1959. Metabolism of cinnamic acid and hydroxycinnamic acid by Lacrob n c i l l ~ r s p a s t o r . i c ~var. t ~ ~ ~cluir~icrrs. s Nature. 184: 14271428.
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Dt,ptrt.ttriettt r~f'Agric~rrltrrrrrl Biolo.qy, Frrc~rItyqfAgriclrlt~rr~, Hokkrrido Utti\~et..sity,S ~ r p ~ ) o rJoO , ~LIII AND
Y. MINO Lnhortrtoty of Ettl~irotttttet~tol Bottrtty, Doprrrtt71c~tttr ~ f A g r o e t ~ ~ ~ i r o t t n tSi.irttce. t ~ t t t r ~ l Obihiro Ut~iversity,Obilziro, Hokkrrirlo, Jtrprrtt Accepted May 7, 1976 HARADA,T . , and Y. M I N O . 1976. Some properties of p-coumar;~te dec;~rboxylase from Cltrdo.s~~orirrtnplilri. Can. J. Microbial. 22: 1258-1262. The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 1 3 "C. respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 "C f o r 5 minand by half at 25 "C in 24 h, but kept almost unchanged at -20 " C a t least for lodays. The activity was not inhibited by potassium cyanide. sodium diethyldithiocarbamate. ethylenediaminetetraacetic acid disodium salt, o r sodium citrate at 10 m M concentration, but was inhibited by p-chloromercuribenzoate o r iodoacetate at 0. I m M , the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid, cinnamic acid, o r p-methoxycinnamic acid, but not by fitmaric acid. acrylic acid, p-hydroxystyrene, furcatin, p-hydroxyphenylacetic acid, or phloretic acid. An ~~nsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. K , value for trrrtrs- p-coumaric acid was a b o ~ l 6.5 t x 10-4 M. HARADAT , . , et Y . M I N O . 1976. Some properties of p-coumarate decarboxylase from Clrrclo.sporirrtitpltlci. Can. J . Microbiol.22: 1258-1262. Le pH et la temperature optimales de la p-coumarate dkcarboxylase sont de 6.0 et 23 "C respectivement. L'activite de I'enzyme est rCduite aux trois quarts par un traitement thermique a 35 "C pendant 5 min et de rnoitie en 24 h a 25 "C; cependant, il n'y a presque pas de changement h -20 "C, pendant au moins I0 jours. L'activite n'est pas inhibee par le cyanure de potassium, le sodium diethyldithiocarrnate. le sel disodique de I'acide ethyltne-diamine-tetrahtique ou le citrate de sodium i~ 10 m M . Toutefois elle est inhibee par le p-chloromercuribenzoate ou I'iodoacetate B 0. I mM: I'inhibition par le p-chloromercuribenzoate est empcchee grandement par la presence du glutathion reduit ou le dithiothreitol. L'activite est inhibee par I'acide maleique, I'acide cinnamique ou I'acide 11-methoxycinnamique, mais ne I'est pas par I'acide fumarique, I'acide acrylique. lep-hydroxystyrene, la furcatine, I'acidep-hydroxyphCnylac6tique ou I'acide phloretique. Un groupement p-hydroxy non-substitue sur I'anneau benzene et une chaine laterale d'acide acrylique sont requis pour I'activite de I'enzyme. La valeur K , pour I'acide tt,trt~s-p-coumariqueest, approximativement. 6.5 x 10-WM. [Traduit par le journal]
Introduction Enzymes decarboxylating cinnamic acid derivatives have been demonstrated to occur in several microorganisms ( I , 3 , 4 , 7). In a previous paper (4), we reported that the timothy leaf spot fungus, Cladospori~inzphlei, has an ability to decarboxylateTPCA2 to p-hydroxystyrene. Some properties of p-coumarate decarboxylase from Aerobacter species have been reported (3), but little is known about the enzymes from fungal sources. This paper described some chemical and physical properties of p-coumarate decarboxylase from C. phlei. 'Received October 14, 1975. 2rrntrs-p-Coumaric acid.
Materials and Methods Flrtignl C~~ltrrre mtrl Enz~jtt~e Prc~pnrrrtiott The timothy leaf spot fungus Clar~osporirut~ plrlei de Vries was used. This fungus was isolated from a local variety of timothy (Pltler~tiipratetrsc, L.) in the Tokachi district in 1969 and identified by Dr. T . Narita (Obihiro University). Stock cultures were kept as slants on the potato agar at 25 " C in the dark for a month. The potato agar slant was prepared as follows. T w o hundred grams o f minced potato tubers were boiled in 1 litre of water for half an hour, followed by filtration through gauze. Twenty grams of glucose and 15 g of pulverized agar were added to the filtrate diluted t o 1 litre with water a n d a 7-ml portion in a test t ~ ~ was b e sterilized at 120°C for 20 min. From this stock culture, a loopful of the fungus was inoculated into a 50-ml erlenmeyer flask containing 20 ml of the liquid medium consisting of 10.0 g glucose, 2.0 g casaniino acid, 1.2 g Na,CO3.10 H 2 0 , 1.0 g KH2P0,, a n d 0.5 g MgS0,.7H20, per litre, a n d 0.5 m g
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HARADA A N D MINO
pyridoxine, 0.1 mg thiamine, 0 2 nig FeS0,.7H20, 0.2 mg ZnSO,.7H,O, and 0.2 mg MnS0, 7 H 2 0 , per litre, and cultured wlthout shaking at 25°C in the dark for 20 days. The liquid medlum was adjusted to pH 6 0 with I N HCl and then sterilized at 120 "C for 20 min before use. The cultured fungus was washed with deionized water. After removing excess water on the surface with filter papers, 20 g of the wet cells were ground thoroughly in 5 ml of 0.1% NaCl solution with 0.5 g of quartz sand in a mortar at 0 "C, followed by centrifugation at 25 000 g at 2 "C for 10 min. Two millllitres of the supernatant solution was chromatographed on a Sephadex G-100 column (1.5 cm x 30 cm) using 0.1 M NaCl solutlon as eluant at a flow rate of 30 ml/h. The eluate was collected In 5-mI fractions. Elutlon profiles of the enzyme and protein are shown in Fig. I. Fractions in tubes Nos. 23-39 were comblned and used as the enzyme solution (0.4 mg of protein/ml). Protein concentration was determined by the method of Lowry et a/. (6) with bovine serum albumln used for a standard.
TUBE N o
FIG. I . Elution profiles of protein (broken line) and enzyme activity (solid line) after Sephadex G-100 column chromatography. The enzyme activity was expressed as the evolution of C 0 2 / I ml of enzyme solution in each fraction per first 30 min.
Assny of the Etrzynle Actiurly The enzyme activlty was determined by measuring the evolution of carbon dloxide during the first 30 min at 20 OC in the reaction mixture consisting of I ml each of enzyme solution containing 0.4 mg of protein and phosphate buffer (67 mM, pH 6.0) in the main chamber and 0.3 ml of 50 niMTPCA (final concentration, 6.5 m M ) in the sidearm, unless otherwise stated. After the manometers were equilibrated for 15 min, the reactlon was initiated by the addition of a substrate from a sidearm. Each measurement was corrected with a proper blank.
Clletnicals D~hydrocaffeicand dihydroferulic acids were prepared from the correspond~ngunsaturated acids, respectively, by hydrogenation in 80% methanol solutlon in the presence of a palladium catalyst. p-Hydroxystyrene was synthesized by the method of Dale and Hennis (2) p-Methoxycinnamic acid was provlded by Mr. M. Terazawa (Obihiro Universlty) and furcatin by Dr. H. Iniazeki (Nagoya Universlty). The other chemicals obtained commercially were used without further ourlfica-
Results Ccr~,bon Dioxide Evolirtion from TPCA by the Enzytlze As shown in Fig. 2, decarboxylation from TPCA proceeded almost linearly during the first 30 min and thereafter decreased gradually until decarboxylation ceased completely after 2 h. pH and Tenzperature The enzyme activity above pH 6.5 was determined by measuring the evolution of total carbon dioxide after acidifying the reaction mixture with 0.2 ml of 1 N H,SO, using a manometer flask with two sidearms. The optimal pH and temperature of the enzyme were around 6.0 (Fig. 3) and 23 "C (Fig. 4), respectively.
30
60
90
120
MINUTE
FIG. 2. Evolution of carbon dioxide from TPCA.
FIG.3. Effect of pH on the enzyme activity. Open and closed circles indicate Mcllvaine (citrate-phosphate) and phosphate buffers, respectively. The enzyme activity was expressed as the evolution of C0,/0.4 mg of protein per first 30 min.
Eflect of Heat Treatment Two millilitres of the enzyme solution in a test tube was shaken (120 rpm) in a water bath at a given temperature for 5 min and then cooled
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CAN. J. MICROBIOL. VOL. 2 2 , 1976
TABLE2. Effect of storage temperature on the enzyme activity
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Rel. % act. Storage period, days
20 @C*
0 "C*
- 20 "C*
NOTE:T h e enzyme activity measured immediately after preparation was e x ~ r e s s e das loo'%.. 15
20
25
30
35
enzyme activity was tested using a Inanometer flask with two sidearms. As shown in Table 3, PCMB3 or iodoacetate at 0.1 mM concentration promptly with cold water. As shown in Table 1, inhibited the activity, but the inhibition by exposure of the enzyme solution to 60°C for PCMB was markedly protected in the presence 5 min brought about the complete disappearance of reduced glutathion or dithiothreitol a t 1 mM. of the activity and the activity was reduced to This suggests that SH groups in the enzyme about three quarters even at 35 "C for 5 min, molecule are required for activity. indicating that the enzyme was comparatively Inhibition by TPCA Analogs heat-labile. The enzyme activity was examined in the Changes in the Etlzyme Activity Stored at Varied presence of a compound related structurally to TPCA. The activity was inhibited by rnaleic, Te17~perat~ires cinnamic, orp-methoxycinnamic acid, but not by The enzyme solution containing each of 1 m M chloramphenicol and dihydrostreptomycin (as fumaric acid, acrylic acid, p-hydroxystyrene, antimicrobial agents) was stored at different furcatin, p-hydroxyphenylacetic acid, or phloretic temperatures for designed periods. As shown in acid. Table 2, the activity was -reduced by half at Substrate Specificify 20 "C and was slightly lost even at 0 "C in 24 h. Substrate specificity was examined using both a non-heat-treated and a heat-treated (35 "C for Effect of Metal Chelatorsand Sulfhydryl Inhibitors The enzyme activity was not inhibited by 5 min) enzyme preparation. The relative decarpotassium cyanide, sodium diethyldithiocarba- boxylation rates are presented in Table 5. mate, ethylenediaminetetraacetic acid disodium Among the compounds tested, cis-p-coumaric, salt, or sodium citrate at 10 m M concentration, bans-p-coumaric, caffeic, and ferulic acids were suggesting that metal ions are not involved in the decarboxylated. These results reflect the fact that activity. The effect of sulfhydryl inhibitors on the an unsubstituted 4-hydroxy group on the benzene ring and an acrylic acid side chain are required for enzyme activity. Decarboxylation TABLE1. Effect of heat-treatment rate was decreased by the introduction of a on the enzyme activity substituent into the 3-position of the benzene Treatment ring. Carbon dioxide evolution by heat-treated temp., "C Rel. % act. enzyme was nearly three quarters of that by nonheat-treated enzyme preparation irrespective of the substrate used, indicating that all compounds decarboxylated are probably attacked by the same enzyme. TEMPERATURE ('C)
FIG.4. Effect of temperature on the enzyme activity.
NOTE: T h e enzyme activity was measured manometrically at 20°C as previously described. T h e activity after a 5-min incubation a t 30°C (88 p1 C02) was expressed as 100%.
K,,, Value The Michaelis constant for TPCA was calcu3p-Chloromercuribenzoate.
HARADA AND MINO
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TABLE3. Effect of SH inhibitors on the enzyme activity SH inhibitor
Rel. % act.
None (TPCA only) Iodoacetate (0.1 m M ) PCMB (0.1 mM) PCMB (0.1 mM) + reduced glutathione (I mM) PCMB (0.1 mM) + dithiothreitol (I mM)
100 32 0 68 78
NOTE:The activity in the absence of any SH inhibitor was expressed as 100%. Immediately after adding a SH compound, PCMB and TPCA were simultaneously added to the enzyme solution. The final concentration of TPCA was 6.5 m M in each case. The concentrations of SH inhibitors and SH compounds were also final ones.
TABLE4. Effect of TPCA analogs Analogs, 5 rnM
Rel. % act.
None (TPCA only) Maleic acid Fumaric acid Acrylic acid p-Hydroxystyrene Furcatin Cinnamic acid p-Methoxycinnarnic acid Phloretic acid
100 82 100 100 100 100 56 72 100
SUBSTRATE I
NOTE:The enzyme acuvlty In the absence of any TPCA analog was expressed as 100Z The solution contalnlng TPCA and an analog, whlch was prevlously adjusted to pH 6.0, was added to the enzyme solut~on. The final concentration of TPCA was 6 5 m M In each case
CONCENTRATION (.10'~)
FIG. 5. K,,,value for TPCA. The value was calculated from a Lineweaver-Burk plot.
lated to be about 6.5 x lop4 M from a Lineweaver-Burk plot as presented in Fig. 5.
Discussion Some chemical and physical properties of pcoumarate decarboxylase from C. phlei were
examined. TPCA decarboxylation by the enzyme proceeded almost linearly during the first 30 min and thereafter decreased gradually (Fig. 2). The enzyme is probably considered to be inactivated in the course of the reaction, since the product p-hydroxystyrene did not inhibit the activity. Though the stability of p-coumarate decarboxylase from other sources has not been reported,
TABLE5. Substrate specificity COz evolut~on
Substrate a c ~ d Cinnamic / I nn~-p-Couniarlc cis-p-Coumaric 111-Coumar~c o-Coumaric Phloretic p-Methoxycinnarnic Caffeic Dihydrocaffeic Ferulic Dihydroferullc Sinapic Acrylic Crotonic
Non-treated (N)
Treated at 35 "C (T)
0 90 140 0 0 0 0 50 0 40 0 0 0 0
0 70 95 0 0 0 0 35 0 30 0 0 0 0
T/N ratio* 0.77 0 68
0.70 0.75
NOTE Figures gl\en are C 0 2(vl) evolved during the first 30 mln F ~ n asubstrate l concentrailon was 6 5 mM. * C 0 2 evolution by heat-treated enzyme/C02 e\olut~onby non-heat-treatcd enzyme.
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1262
CAN. J. MlCROBlIOL. VOL. 22. 1976
the C , phlei enzyme seems to be fairly labile, since the enzyme was slightly inactivated even by heat treatment at 35 "C (Table I) and reduced by half at 25 "C in 24 h after enzyme preparation (Table 2). Furthermore, treatment by ammonium sulfate and chromatography on diethylaminoethyl (DEAE)-cellulose or Dowex 50 brought about its complete inactivation and considerable activity was lost during dialysis against tap water (data are not detailed here). This lability may be explained in terms of the oxidation of sulfhydryl groups required for the activity (Table 3). Cladospori~in~ plilei decarboxylase was inhibited by maleic acid, but not by fumaric acid as was the case for Aerobacter enzyme (3). Whiting and Carr (7) reported the bacterial decarboxylation of TPCA through a reductive route involving a hydroxyphenylpropionic acid as an intermediate. In contrast with their results, the decarboxylation of TPCA by C. phlei proceeds without any modification of the double bond in the acrylic side chain (4). The formation of styrene from cinnamic acid by some molds (1, 5) has been reported. The enzyme from C. phlei is considered to be different from those enzymes, judging from the results presented in Table 5. Aerobacter decarboxylase ( 3 ) was reported to require a relatively unhindered 4hydroxy group on the aromatic ring and an acrylic side chain. A similar situation appears to
be the case for the C , plzlei enzyme. In this study, enzyme purification has been unsuccessful because of instability. The problems mentioned above remain to be clarified by further purified enzyme.
Acknowledgments We are indebted to Prof. Y. Nishijima for reading this manuscript. We also thank Miss Y. Nabara for her technical assistance.
I. CLIFFORD, D. R., J. K. F A U L K E R J., R. L. WALKER, and D. WOODCOCK. 1969. Metabolism of cinnamic acid by Aspergilllrs niger. Phytochemistry. 8: 549-552. 2. DALE,W. J., and H. E. HENNIS.1958. Substituted styrenes, 111. The syntheses and somechemical properties of vinylphenols. J. Am. Chem. Soc. 80: 3645-3649. , J., J. C. L E W I SJ. , W. CORSE,and R . E. 3. F I N K L EB. L U N D I N1962. . Enzyme reactions with phenolic compounds: formation of hydroxystyrenes thro~lghthe decarboxylation of 4-hyd~.oxycinnamic acids by Aerobtrcrer. J. Biol. Chem. 237: 2926-293 1. 4. HARADA.T., and Y. MINO. 1973. Formation of 4-hydroxystyrene frornp-coumaricacid by the timothy leaf spot fungus, Cltrtlospor-ilrm phlei. Ann. Phytopathol. Soc. Japan, 39: 438-440. 5. JAMINET,F. 1950. Hydrocarbonic fermentation in a tolu sirup mixture. J. Pharm. Belg. 5: 191-201. 0 . H.. N . J. ROSEBROUGH, A . L. FARR,and R. 6. LOWRY, J. RANDALL. 1951. Protein measurementwith the Foiin phenol reagent. J. Biol. Chem. 193: 265-275. 7. WHITING, G. C., and J. G. CARR.1959. Metabolism of cinnamic acid and hydroxycinnamic acid by Lacrob n c i l l ~ r s p a s t o r . i c ~var. t ~ ~ ~cluir~icrrs. s Nature. 184: 14271428.
