- Email: [email protected]
Identification, Properties and Genetic Control of p-CoumaroylCoenzyme A, 3-Hydroxylase Isolated from Petals of Silene dioica
Identification, Properties and Genetic Control of p-CoumaroylCoenzyme A, 3-Hydroxylase Isolated from Petals of Silene dioica JOHN KAMSTEEG, JAN VAN BREDERODE, PAUL M. VERSCHUREN and GERRIT VAN NIGTEVECHT Department of Home Economics, Household Technology Lab., Agricultural University, Ritzema Bosweg 32a, NL-6703 AZ Wageningen, The Netherlands and Department of Population and Evolutionary Biology, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. Received January 27, 1981 . Accepted February 4, 1981
Summary An enzyme catalyzing the hydroxylation of the 3-position of p-coumaroyl-Coenzyme A has been -demonstrated in petal extracts of Silene dioica plants. For optimal activity NADPH, FAD, and molecular oxygen are necessary. The hydroxlating activity is governed by gene Pj in pink petals of pip plants this activity was absent. In vivo gene P controls both the hydroxylation pattern of the anthocyanin B-ring and that of the acyl group. The enzyme can use p-coumaric acid as non-substrate effector. In this case hydrogen peroxide is formed and p-coumaric acid remains unchanged during the course of the reaction. Although the enzyme isolated from petals of pip plants was not able to hydroxylate pcoumaroyl-CoA, it could still use p-coumaric acid as non-substrate effector.
Key words: Silene dioica, Caryophyllaceae, cyanidin-, pelargonidinglucosides, hydroxylation, p-coumaroyl-CoA, genetic control, anthocyanin biosynthesis.
Introduction One of the most intriguing and still unresolved problems in flavonoid biosynthesis is the exact location of the stage at which the hydroxylation pattern of the Bring is determined and the mechanism which is involved in this hydroxylation reaction. According to the «cmnamIC acid starter» hypothesis of Hess (1964, 1967 and 1968), the substitution pattern of the B-ring is determined at the cinnamic acid stage, and persists throughout the biosynthetic sequence to produce the corresponding flavonoid. Grisebach (1973), however, demonstrated that elaboration of the hydroxylation pattern of the B-ring of flavonoids can also occur at chalcone/ flavanone- and even later stages of the flavonoid pathway. In a previous paper (Kamsteeg et ai., 1980), it has been demonstrated that in
Silene dioica the hydroxylation pattern of the anthocyanin B-ring is determined
z.
PJlanzenphysiol. Ed. 102. S. 435-442.1981.
436
JOHN KAMSTEEG
et al.
at the p-coumaroyl-CoA stage. The enzyme, p-coumaroyl-CoA, 3-hydroxylase which catalyzes the conversion of p-coumaroyl-CoA to caffeoyl-CoA is governed by gene P. When (f3_14C) p-coumaroyl-CoA was incubated with a protein preparation of petals of PIP plants, in the presence of FAD and NADPH, radioactivity was incorporated into caffeoyl-CoA; with a petal extract of pip plants no caffeoyl-CoA was formed. In this paper we describe the properties of the flavoprotein controlled by gene P, which catalyzes the hydroxylation of p-coumaroyl-CoA.
Material and Methods Plant material Silene dioica was grown
In the experimental garden of the Department of Population and Evolutionary Biology of Utrecht University. The growing conditions and crossing methods were the same as published before (van Nigtevecht, 1966). The genotypes were obtained by means of selection and inbreeding (Kamsteeg et al., 1976). The petals of opening flowers of both PIP and pip plants were collected in tubes placed in crushed ice and stored at -17°C.
Chemicals p-Coumaroyl-CoA was a kind gift of Dr. R. Siitfeld. All other chemicals were supplied by Boehringer, Mannheim, Germany. Carbon monoxide was synthesized by the addition of formic acid to warm concentrated sulphuric acid as described by Parkes (1963).
Enzyme preparation All operations were carried out between 0 and 4°C. Five grams of petals were homogenized in an all glass Potter Elvehjem homogenizer in 2 times their weight of a 20 mM p-mercaptoethanol, 5 Ofo soluble polyvinylpyrrolidone, 0.1 % Triton X-IOO, 50 mM potassiumsodium phosphate buffer (pH 7.8), and centrifuged for 30 minutes at 38,000 Xg. The supernatant fraction was either passed through a Dowex IX2 (0.8 by 10 cm) column or a Polyclar AT (PVP, I by 30 cm) column. The eluates were if necessary concentrated in an Amicon on-line concentrator (CEC I) with an UM-IO filter (Amicon, Lexington, Massachusetts), and subsequently passed through a Sephadex G-150 column (2.5 by 40 cm). The columns were equilibrated and eluted with a 4 mM p-mercaptoethanol, 10 mM potassium-sodium phosphate buffer (pH 8.0). The pooled peak Sephadex G-150 fractions, corresponding to a molecular weight of 30,000 daltons, were used in tests to determine the enzyme properties.
Enzyme assays The enzyme was assayed: 1. by measuring the p-coumaroyl-CoA dependent NADPH oxidation at 340 nm. The reaction mixture contained in a total volume of 1.2 ml: 0.1 ,uMol NADPH, 10 nMol FAD, O.I,uMol substrate p-coumaroyl-CoA or the non-substrate effector p-coumaric acid, 8 flMol potassium-sodium phosphate buffer (pH 8.0), 3.2,uMol p-mercaptOethanol, and 0.8 ml enzyme (Absorption coefficient of NADPH at 340 nm f 6,220). 2. By following the p-coumaroyl-CoA dependent reduction of FAD at 450 nm. The reaction mixture contained in a tOtal volume of 1.2 ml: 40 nMol FAD, 50 nMol NADPH, 50 nMol substrate p-coumaroyl-CoA or the non-substrate effectOr p-coumaric acid,
Z. Pjlanzenphysiol. Ed. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
437
8,uMol potassium-sodium phosphate (pH 8.0), 3.2,uMol p-mercaptoethanol, and 0.8 ml enzyme (Absorption coefficient of FAD at 450 nm c 11,300). 3. By measuring the oxygen consumption. The oxygen consumption was measured in a reaction vessel of a model 53 biological oxygen monitor with a 5301 standard bath assembly (Yellow Springs Instruments Co., Yellow Spring, Ohio), which contained in a total volume of 3.5 ml: 4 flMol NADPH, 0.38,uMol FAD, 1.5 ,uMol substrate pcoumaroyl-CoA or non-substrate effector p-coumaric acid, 10,uMol potassium-sodium phosphate (pH 8.0), 4 fiMol p-mercaptoethanol, and 1 ml enzyme. The reaction was started from an air equilibrated solution by the addition of substrate and was recorded for 5 minutes.
Reduction
0/ the enzyme
The enzyme (complexed with FAD and substrate or uncomplexed) was reduced: (i) under nitrogen by the light irra-dition of the enzyme solution in the presence of 20 mM EDTA according to the method of Spector and Massey (1972). (ii) By the addition of crystals sodium dithionite to the enzyme solution under anaerobic conditions. (iii) By the addition of an excess NADPH to the enzyme solution under anearobic conditions.
Protein assays The protein content was determined with the Bio-Rad protein assay mixture (Bio-Rad Laboratories, Richmond, California) and according to the metho-d of Lowry et al. (1951).
Results
Enzyme properties Incubation of p-coumaroyl-CoA with a crude petal homogenate of PIP Silene dioica plants, in the presence of NADPH and molecular oxygen, resulted in the formation of caffeoyl-CoA, with a corresponding decrease of NADPH and molecular oxygen. After centrifugation for 30 minutes at 38,000 X g this hydroxylating activity was mainly present in the supernatant (Table 1). Treatment with Dowex 1X2 or precipitation with solid ammonium sulphate led to a considerable loss in Table I: Purification of p-coumaroyl-CoA, 3-hydroxylase from petals of PIP Silene dioica plants. Protein fraction
Homogeniud crude extract Supernatant crude extract Supernatant 38,000 x g Precipitate 38,000 x g Dowex 1 X 2 eluate Sephadex G-150 eluate
Volume (ml)
14.3 12.7 11.6 1.0 17.0 16.8
,:. One unit is the amount of enzyme which assay mixture (see Material and Methods).
Protein (mg/ml)
6.3 4.6 4.4 1.5 2.15 0.36 con~umes I
Total
Specific
lCtlvlty
activity
(units''Imin)
(units/ nlin/1l1g prot. )
973 713 618 5.S 2550 1510
Enz\'me pUrIn
10.~
11.1 11.1 1.3 70 -117
1.13 1.12 0.11 6.5 38.6
nmol oxvgen per minute al 10°C in the standard
Z. P/lanzenphysiol. Bd. 102. S. 435-442. 1981.
438
JOHN KAMSTEEG
e{ al.
activity, which could be restored by the addition of FAD to the assay. This sugge5'ts that FAD is also necessary for enzymatic activity. As with Sephadex G-25 chromatography of the 38,000 X g supernatant this loss in activity was not perceptable, FAD is probably bound to the enzyme. By treatment with Dowex lX2 and Sephadex G-150 chromatography the enzyme was purified fourtyfold (Table 1). The pooled Sephadex G-150 fractions corresponding to a molecular weight of 25-40,000 daltons were routineously used for the investigation of the enzyme properties. With p-coumaroyl-CoA as substrate the enzyme showed a strict requirement for NADPH. Nevertheless when p-coumaric acid was used as non-substrate effector NADH could serve as reducwr to. In this case the enzyme showed a four times higher oxygen consumption with NADPH than with NADH. The in vitro by sodium dithionite or photochemically by EDTA reduced enzyme showed oxygen consumption in the presence of substrate as well. tJ-Mercaptoethanol has a stabilizing effect on the enzyme. The enzyme is strongly inhibited by sulfhydryl specific reagents. At a concentration of 10- 4 M, p-chloromercuribenzoate completely inhibited the reaction. This inhibition and the inhibition by Zn 2+ could be largely reversed by the addition of cysteine or p-mercaptoethanol. The recovered activities were 75 Ofo and 95 %, respectively. The enzyme was irreversibly inhibited by HgCI 2 • The divalent metal ions Ca 2+, Co 2+, Mg2+ and Mn 2+ did not stimulate the reaction rate nor did EDTA. The respiratory poisons potassium cyanide and sodium azide (10- 3 M) did not inhibit the enzyme. Potassium ferricyanide inhibited the oxygen consumption completely. This compound acts as electron acceptor; both under aerobic and anaerobic conditions the reduction of potassium ferricyanide could be demonstrated by following the absorption at 420 nm (HOWELL et aI., 1972). According to the criteria of Massey and Hemmerich (1975) the enzyme can be classified as a mixed function oxidase or mono-oxygenase. The flavoprotein incorporates one atom of molecular oxygen into the substrate to yield an oxygenated, in this case hydroxyla'ted product. Many mono-oxygenases possess oxidase activity as well. This oxidase activity can be elicited by so-called effectors. These effectors cause the reduction of FAD by NADPH, but remain unchanged during the course of the reaction. The reduced enzyme reacts with molecular oxygen to yield hydrogen peroxide. In Our assay the oxydation of NADPH and concomitant molecular oxygen consumption without hydroxylation were demonstrated with the compounds pcoumaric acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. That hydrogen peroxide is formed as well, can be demonstrated by the addition of catalase, which liberates again half the amount of molecular oxygen consumed, compared to a test system in which the possible endogenous catalase was inhibited by the addition of sodium azide (Fig. 1 B). With ·the substrate p-coumaroyl-CoA, the amount of molecular oxygen consumed in the assay to which sodium azide had been added and Z. Pflanzenphysiol. Bd. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
B
A
100
----.~
----.
25
\ \
---..
\
'. ;:
3
'-
TIME(MINI
\
\
z
\
0
I-
2 \
cf2 '-" 80
25
\
\
« a:
439
\ \
2
3
7 '---'::-T~IM':":E'-;-(M:-:-C:1NJ - l
,,
,,
\
60
,,
,,
:::J
I-
«
(f)
z
\
\
40
\ \
w
\
\
<.:)
\
>-
x 20
0
0
2
4
6
2
4
6
TIME (MINUTES) Fig. 1: The time course of oxygen consumption in the presence of 0.5 mM sodium azide (--) is compared to catalase (---). At the position of the arrow in A, the substrate p-coumaroyl-CoA and in B the effector p-coumaric acid were added. Inset: the NADPH oxidation at 340 nm (see Material and Methods).
the amount of oxygen consumed in the presence of catalase were about equal (Fig. 1 A). Carbon monoxide and sodium cholate, inhibitors of cytochrome P-4S0, had no influence on the oxidase activity nor on the hydroxylating activity of the enzyme.
Genetic control The data in Table 2 show that the dominant allele of gene P is necessary for the formation of the enzyme that catalyzes the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA. No p-coumaroyl-CoA hydroxylating activity was found in petals of pip plants. The oxidase activity however, is still retained in petals of plants homozygous recessive for gene P (Table 2).
pH optimum Both in glycylglycine and in phosphate buffer the enzyme has a fairly narrow pH range. In glycylglycine buffer it exhibits maximal activity at pH 8.6; in phosphate buffer this value is lower (pH 8.1). The standard assays were performed in phosphate buffer at pH 8.0.
z.
Pjlanzenphysiol. Ed. 102. S. 435-442. 1981.
440
JOHN KAMSTEEG
et al.
Table 2: Genetic control of NADPH: p-coumaroyl-CoA, 3-hydroxylase activity in petals of S. dioica plants. Genotype
Enzymatic activity':') with effector p-coumaric acid
Enzymatic activity':) with substrate p-coumaroyl-CoA
PIP
323
386
pip
141
none
,:.) The activity was determined in the Sephadex G-tSO fractions (M. W. 30,000) and is expressed as the oxygen consumption in the standard assay at 20°C in nMollmin/mg protein.
Kinetics Michaelis constants for the hydroxylMion reaction were determined by means of linear regression analysis on the Eadie-Hofstee linear transformation Cs/V versus Cs (Dowd and Riggs, 1965). The Km for p-coumaroyl-CoA was 0.2,uM. For the part in which a linear relationship between [FAD]/V and [FAD] was observed a Km was calculated of 1,uM for FAD. A Km of O.l,uM was found for NADPH. Maximal activity was obtained with substrate concentrations of about 10-4 M; at concentrations above 10- 3 M the reaction rate was inhibited. Other effectors or aromatic compounds at concentrations higher than 10- 3 M are non-competitive inhibitors. Also free Coenzyme A inhibited the reaction rate strongly.
Molecular weight determination The molecular weight of the enzyme was determined with a Sephadex G-150 column calibrated with cytOchrome c, chymotrypsinogen A, egg albumin, aldolase, and catalase as standards (Andrews, 1965). In the fractions corresponding with a molecular weight of 30,000, 60,000 and 120,000 dalton a maximum in hydroxylating activity was detected. The oxygen consumption in the 60,000 and 120,000 fraction was higher than in the 30,000 dalton fraction, but the oxygen consumption in these two fractions was only linear for a very short period. Dowex 1X2 treatment before Sephadex G-150 chromatography caused an increase of activity in the 30,000 dalton fraction. Without this step most of the activity was present in the 120,000 dalton fraction. It is remarkable that no peak in enzyme activity was detected which corresponded with a molecular weight of 90,000 daltons.
Spectrophotometric investigations The difference spectrum of the enzyme reduced with sodium dithionite in the presence and absence of carbon monoxide showed a photOdissociable peak at 450 nm, which is typical for cytochrome P-450 (Omura and SatO, 1964; Ambike et al., 1970). Additional evidence for the presence of cytochrome P-450 Z. PJlanzenphysiol. Bd. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
441
was obtained from the appearance of an absorption peak at 421 nm in the difference spectra of the enzyme with either 1 mM p-chloromercuribenzoate or 4 Ofo sodium cholate. These reagents catalyze the conversion of cytochrome P-450 to cytochrome P-420 (Cooper et aI., 1964; Ichikawa and Yamaro, 1967). Discussion In this paper we describe an enzyme isolated from petals of PIP Silene dioica plants, which catalyzes the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA. This reaction is NADPH-dependent and uses molecular oxygen. The enzyme-bound FAD can be removed by Dowex 1X2 treatment or by precipitation with solid ammonium sulphate, but not by gel filtration. According to the criteria of Massey and Hemmerich (1975) this enzyme is a flavoprotein mono-oxygenase; NADPH is necessary for catalytic activity, the hydroxylation of an aromatic compound is catalyzed, and more important, p-coumaroyl-CoA is b(}th effector and substrate. In the presence of only NADPH there is no reduction of the enzyme-bound FAD. Addition of substrate p-coumaroyl-CoA causes an enormous stimulation of the NADPH oxidation. The effector role of the substrate can be simulated by substrate analogues which also increase the rate of reduction of the enzyme bound flavin by NADPH, but cannot serve as hydroxylatable substrates. The compounds, p-coumaric acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde, caused NADPH oxidation in our assay system. The reduction product of molecular oxygen in these cases is hydrogen peroxide rather than water, the product normally formed with hydroxylatable substrates. This could be demonstrated by the addition of catalase, which in the case of hydrogen peroxide formation, liberated again half the amount of molecular oxygen consumed. With the substrate p-coumaroyl-CoA no oxygen liberation could be detected after the addition of catalase to the assay. The compounds mentioned above therefore fulfill the conditions postulated by Massey and Hemmerich (1975) for non-substrate effectors. In plants homozygous recessive for gene P the p-coumaroyl-CoA hydroxylating activity is lost. So gene P is the structural gene for p-coumaroyl-CoA hydroxylation. Remarkably the oxidase activity is retained in homozygous recessive pip plants. With p-coumaric acid as non-substrate effecwr, hydrogen peroxide is still formed. Although there are strong indications that cytochrome P-450 is present in the purified Sephadex G-150 fractions which correspond with a molecular weight of 25-40,000 daltons, it is probably not involved in the hydroxylation mechanism. Poisening of cytochrome P-450 with carbon monoxide or other compounds had no influence on the reaction rate. We therefore suggest that the hydroxylation of p-coumaroyl-CoA occurs directly at the enzyme-bound FAD. Acknow ledgemen ts The authors are much indebted to Dr. R. Sutfeld, Botanisches Institut der Westfalischen Wilhelms-Universitat, Munster, Germany, for kindly supplying a sample of p-coumaroyl-
z. Pflanzenphysiol.
Bd. 102. S. 435-442. 1981.
442
JOHN KAMSTEEG et al.
CoA. We wish to thank Mr. George E. W. Thorig for his helpful remarks and Mr. D. Smit for preparing the illustration.
References AMBIKE, S. H., R. M. BAXTER, and N. D. ZAHID: The relationship of cytochrome P-450 levels and alkaloid synthesis in Claviceps purpurea. Phytochemistry 9, 1953-1958 (1970). ANDREWS, P.: The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606 (1965). DOWD, J. E. and D. S. RIGGS: A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations. J. Bioi. Chern. 240, 863-869 (1965). ENTSCH, B., D. P. BALLOU, and V. MASSEY: Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase. J. BioI. Chern. 251, 2550-2563 (1976.) GRISEBACH, H.: Comparative biosynthetic pathways in higher plants. In: SWAIN, T., (Ed.): Chemistry in evolution and systematics pp. 487-511. Butterworths, London, 1973. HESS, D.: Der Einbau Methylgruppen-Markierter Ferulasaure und Sinapinsaure in die Anthocyane von Petunia hybrida. Planta 60, 568-581 (1964). - Die Wirkung von Zimtsauren auf die Anthocyansynthese in isolierter Petalen von Petunia hybrida. Z. Pflanzenphysiol. 56, 12-19 (1967). - Biochemische Genetik: eine Einfiihrung unter besonderer Beriicksichtigung hoherer Pflanzen. In: HESS, D., (Ed.): Phenole und Phenolderivate pp. 23-29. Springer-Verlag, Berlin, 1968. HOWELL, L. G., T. SPECTOR, and V. MASSEY: Purification and properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. J. Bioi. Chern. 247, 4340-4350 (1972). ICHIKAWA, T. and T. YAMANO: Reconversion of detergent- an{] sulfhydryl reagent-produced P-420 to P-450 by polyols and glutathione. Biochim. Biophys. Acta. 131,490-497 (1967). KAMSTEEG, J., J. VAN BREDERODE, and G. VAN NIGTEVECHT: Pleiotropic effect of a pelargonidin-hydroxylation gene in Silene dioica? Phytochemistry 15, 1917-1918 (1976). - - - Genetical and biochemical evidence that the hydroxylation pattern of the anthocyanin B-ring in Silene dioica is determined at p-coumaroyl-CoA stage. Phytochemistry 19,1459-1462 (1980). LOWRY, O. H., H. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MASSEY, V. and P. HEMMERICH: Flavin and Pteridine mono-oxygenases. In: BOYER, P. D. (Ed.): The enzymes Vol. XII. part B. Oxidation-rwuction, electron transfer (II), oxygenases and oxidases (I). pp. 191-253. Academic Press, New York, 1975. NIGTEVECHT, G. VAN: Genetic studies in dioecious Melandrium. I. Sexlinked and sex-influenced inheritance in Melandrium album and Melandrium dioicum. Genetica 37, 281306 (1966). OMURA, T. and R. SATO: The carbon monoxide-binding pigment of liver microsomes. J. Bioi. Chern. 239, 2370-2378 (1964). PARKES, G. D.: Mellor's modern inorganic chemistry. lIth w. pp. 397-399. Longmans, Green and Co. Ltd., London (1963). SPECTOR, T. and V. MASSEY: Studies on the effector specificity of p-hydroxybenzoate hydrocylase from Pseudomonas fluorescens. J. BioI. Chern. 247, 4679-4687 (1972).
Z. Pflanzenphysiol. Bd. 102. S. 435-442. 1981.
Summary An enzyme catalyzing the hydroxylation of the 3-position of p-coumaroyl-Coenzyme A has been -demonstrated in petal extracts of Silene dioica plants. For optimal activity NADPH, FAD, and molecular oxygen are necessary. The hydroxlating activity is governed by gene Pj in pink petals of pip plants this activity was absent. In vivo gene P controls both the hydroxylation pattern of the anthocyanin B-ring and that of the acyl group. The enzyme can use p-coumaric acid as non-substrate effector. In this case hydrogen peroxide is formed and p-coumaric acid remains unchanged during the course of the reaction. Although the enzyme isolated from petals of pip plants was not able to hydroxylate pcoumaroyl-CoA, it could still use p-coumaric acid as non-substrate effector.
Key words: Silene dioica, Caryophyllaceae, cyanidin-, pelargonidinglucosides, hydroxylation, p-coumaroyl-CoA, genetic control, anthocyanin biosynthesis.
Introduction One of the most intriguing and still unresolved problems in flavonoid biosynthesis is the exact location of the stage at which the hydroxylation pattern of the Bring is determined and the mechanism which is involved in this hydroxylation reaction. According to the «cmnamIC acid starter» hypothesis of Hess (1964, 1967 and 1968), the substitution pattern of the B-ring is determined at the cinnamic acid stage, and persists throughout the biosynthetic sequence to produce the corresponding flavonoid. Grisebach (1973), however, demonstrated that elaboration of the hydroxylation pattern of the B-ring of flavonoids can also occur at chalcone/ flavanone- and even later stages of the flavonoid pathway. In a previous paper (Kamsteeg et ai., 1980), it has been demonstrated that in
Silene dioica the hydroxylation pattern of the anthocyanin B-ring is determined
z.
PJlanzenphysiol. Ed. 102. S. 435-442.1981.
436
JOHN KAMSTEEG
et al.
at the p-coumaroyl-CoA stage. The enzyme, p-coumaroyl-CoA, 3-hydroxylase which catalyzes the conversion of p-coumaroyl-CoA to caffeoyl-CoA is governed by gene P. When (f3_14C) p-coumaroyl-CoA was incubated with a protein preparation of petals of PIP plants, in the presence of FAD and NADPH, radioactivity was incorporated into caffeoyl-CoA; with a petal extract of pip plants no caffeoyl-CoA was formed. In this paper we describe the properties of the flavoprotein controlled by gene P, which catalyzes the hydroxylation of p-coumaroyl-CoA.
Material and Methods Plant material Silene dioica was grown
In the experimental garden of the Department of Population and Evolutionary Biology of Utrecht University. The growing conditions and crossing methods were the same as published before (van Nigtevecht, 1966). The genotypes were obtained by means of selection and inbreeding (Kamsteeg et al., 1976). The petals of opening flowers of both PIP and pip plants were collected in tubes placed in crushed ice and stored at -17°C.
Chemicals p-Coumaroyl-CoA was a kind gift of Dr. R. Siitfeld. All other chemicals were supplied by Boehringer, Mannheim, Germany. Carbon monoxide was synthesized by the addition of formic acid to warm concentrated sulphuric acid as described by Parkes (1963).
Enzyme preparation All operations were carried out between 0 and 4°C. Five grams of petals were homogenized in an all glass Potter Elvehjem homogenizer in 2 times their weight of a 20 mM p-mercaptoethanol, 5 Ofo soluble polyvinylpyrrolidone, 0.1 % Triton X-IOO, 50 mM potassiumsodium phosphate buffer (pH 7.8), and centrifuged for 30 minutes at 38,000 Xg. The supernatant fraction was either passed through a Dowex IX2 (0.8 by 10 cm) column or a Polyclar AT (PVP, I by 30 cm) column. The eluates were if necessary concentrated in an Amicon on-line concentrator (CEC I) with an UM-IO filter (Amicon, Lexington, Massachusetts), and subsequently passed through a Sephadex G-150 column (2.5 by 40 cm). The columns were equilibrated and eluted with a 4 mM p-mercaptoethanol, 10 mM potassium-sodium phosphate buffer (pH 8.0). The pooled peak Sephadex G-150 fractions, corresponding to a molecular weight of 30,000 daltons, were used in tests to determine the enzyme properties.
Enzyme assays The enzyme was assayed: 1. by measuring the p-coumaroyl-CoA dependent NADPH oxidation at 340 nm. The reaction mixture contained in a total volume of 1.2 ml: 0.1 ,uMol NADPH, 10 nMol FAD, O.I,uMol substrate p-coumaroyl-CoA or the non-substrate effector p-coumaric acid, 8 flMol potassium-sodium phosphate buffer (pH 8.0), 3.2,uMol p-mercaptOethanol, and 0.8 ml enzyme (Absorption coefficient of NADPH at 340 nm f 6,220). 2. By following the p-coumaroyl-CoA dependent reduction of FAD at 450 nm. The reaction mixture contained in a tOtal volume of 1.2 ml: 40 nMol FAD, 50 nMol NADPH, 50 nMol substrate p-coumaroyl-CoA or the non-substrate effectOr p-coumaric acid,
Z. Pjlanzenphysiol. Ed. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
437
8,uMol potassium-sodium phosphate (pH 8.0), 3.2,uMol p-mercaptoethanol, and 0.8 ml enzyme (Absorption coefficient of FAD at 450 nm c 11,300). 3. By measuring the oxygen consumption. The oxygen consumption was measured in a reaction vessel of a model 53 biological oxygen monitor with a 5301 standard bath assembly (Yellow Springs Instruments Co., Yellow Spring, Ohio), which contained in a total volume of 3.5 ml: 4 flMol NADPH, 0.38,uMol FAD, 1.5 ,uMol substrate pcoumaroyl-CoA or non-substrate effector p-coumaric acid, 10,uMol potassium-sodium phosphate (pH 8.0), 4 fiMol p-mercaptoethanol, and 1 ml enzyme. The reaction was started from an air equilibrated solution by the addition of substrate and was recorded for 5 minutes.
Reduction
0/ the enzyme
The enzyme (complexed with FAD and substrate or uncomplexed) was reduced: (i) under nitrogen by the light irra-dition of the enzyme solution in the presence of 20 mM EDTA according to the method of Spector and Massey (1972). (ii) By the addition of crystals sodium dithionite to the enzyme solution under anaerobic conditions. (iii) By the addition of an excess NADPH to the enzyme solution under anearobic conditions.
Protein assays The protein content was determined with the Bio-Rad protein assay mixture (Bio-Rad Laboratories, Richmond, California) and according to the metho-d of Lowry et al. (1951).
Results
Enzyme properties Incubation of p-coumaroyl-CoA with a crude petal homogenate of PIP Silene dioica plants, in the presence of NADPH and molecular oxygen, resulted in the formation of caffeoyl-CoA, with a corresponding decrease of NADPH and molecular oxygen. After centrifugation for 30 minutes at 38,000 X g this hydroxylating activity was mainly present in the supernatant (Table 1). Treatment with Dowex 1X2 or precipitation with solid ammonium sulphate led to a considerable loss in Table I: Purification of p-coumaroyl-CoA, 3-hydroxylase from petals of PIP Silene dioica plants. Protein fraction
Homogeniud crude extract Supernatant crude extract Supernatant 38,000 x g Precipitate 38,000 x g Dowex 1 X 2 eluate Sephadex G-150 eluate
Volume (ml)
14.3 12.7 11.6 1.0 17.0 16.8
,:. One unit is the amount of enzyme which assay mixture (see Material and Methods).
Protein (mg/ml)
6.3 4.6 4.4 1.5 2.15 0.36 con~umes I
Total
Specific
lCtlvlty
activity
(units''Imin)
(units/ nlin/1l1g prot. )
973 713 618 5.S 2550 1510
Enz\'me pUrIn
10.~
11.1 11.1 1.3 70 -117
1.13 1.12 0.11 6.5 38.6
nmol oxvgen per minute al 10°C in the standard
Z. P/lanzenphysiol. Bd. 102. S. 435-442. 1981.
438
JOHN KAMSTEEG
e{ al.
activity, which could be restored by the addition of FAD to the assay. This sugge5'ts that FAD is also necessary for enzymatic activity. As with Sephadex G-25 chromatography of the 38,000 X g supernatant this loss in activity was not perceptable, FAD is probably bound to the enzyme. By treatment with Dowex lX2 and Sephadex G-150 chromatography the enzyme was purified fourtyfold (Table 1). The pooled Sephadex G-150 fractions corresponding to a molecular weight of 25-40,000 daltons were routineously used for the investigation of the enzyme properties. With p-coumaroyl-CoA as substrate the enzyme showed a strict requirement for NADPH. Nevertheless when p-coumaric acid was used as non-substrate effector NADH could serve as reducwr to. In this case the enzyme showed a four times higher oxygen consumption with NADPH than with NADH. The in vitro by sodium dithionite or photochemically by EDTA reduced enzyme showed oxygen consumption in the presence of substrate as well. tJ-Mercaptoethanol has a stabilizing effect on the enzyme. The enzyme is strongly inhibited by sulfhydryl specific reagents. At a concentration of 10- 4 M, p-chloromercuribenzoate completely inhibited the reaction. This inhibition and the inhibition by Zn 2+ could be largely reversed by the addition of cysteine or p-mercaptoethanol. The recovered activities were 75 Ofo and 95 %, respectively. The enzyme was irreversibly inhibited by HgCI 2 • The divalent metal ions Ca 2+, Co 2+, Mg2+ and Mn 2+ did not stimulate the reaction rate nor did EDTA. The respiratory poisons potassium cyanide and sodium azide (10- 3 M) did not inhibit the enzyme. Potassium ferricyanide inhibited the oxygen consumption completely. This compound acts as electron acceptor; both under aerobic and anaerobic conditions the reduction of potassium ferricyanide could be demonstrated by following the absorption at 420 nm (HOWELL et aI., 1972). According to the criteria of Massey and Hemmerich (1975) the enzyme can be classified as a mixed function oxidase or mono-oxygenase. The flavoprotein incorporates one atom of molecular oxygen into the substrate to yield an oxygenated, in this case hydroxyla'ted product. Many mono-oxygenases possess oxidase activity as well. This oxidase activity can be elicited by so-called effectors. These effectors cause the reduction of FAD by NADPH, but remain unchanged during the course of the reaction. The reduced enzyme reacts with molecular oxygen to yield hydrogen peroxide. In Our assay the oxydation of NADPH and concomitant molecular oxygen consumption without hydroxylation were demonstrated with the compounds pcoumaric acid, p-hydroxybenzoic acid and p-hydroxybenzaldehyde. That hydrogen peroxide is formed as well, can be demonstrated by the addition of catalase, which liberates again half the amount of molecular oxygen consumed, compared to a test system in which the possible endogenous catalase was inhibited by the addition of sodium azide (Fig. 1 B). With ·the substrate p-coumaroyl-CoA, the amount of molecular oxygen consumed in the assay to which sodium azide had been added and Z. Pflanzenphysiol. Bd. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
B
A
100
----.~
----.
25
\ \
---..
\
'. ;:
3
'-
TIME(MINI
\
\
z
\
0
I-
2 \
cf2 '-" 80
25
\
\
« a:
439
\ \
2
3
7 '---'::-T~IM':":E'-;-(M:-:-C:1NJ - l
,,
,,
\
60
,,
,,
:::J
I-
«
(f)
z
\
\
40
\ \
w
\
\
<.:)
\
>-
x 20
0
0
2
4
6
2
4
6
TIME (MINUTES) Fig. 1: The time course of oxygen consumption in the presence of 0.5 mM sodium azide (--) is compared to catalase (---). At the position of the arrow in A, the substrate p-coumaroyl-CoA and in B the effector p-coumaric acid were added. Inset: the NADPH oxidation at 340 nm (see Material and Methods).
the amount of oxygen consumed in the presence of catalase were about equal (Fig. 1 A). Carbon monoxide and sodium cholate, inhibitors of cytochrome P-4S0, had no influence on the oxidase activity nor on the hydroxylating activity of the enzyme.
Genetic control The data in Table 2 show that the dominant allele of gene P is necessary for the formation of the enzyme that catalyzes the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA. No p-coumaroyl-CoA hydroxylating activity was found in petals of pip plants. The oxidase activity however, is still retained in petals of plants homozygous recessive for gene P (Table 2).
pH optimum Both in glycylglycine and in phosphate buffer the enzyme has a fairly narrow pH range. In glycylglycine buffer it exhibits maximal activity at pH 8.6; in phosphate buffer this value is lower (pH 8.1). The standard assays were performed in phosphate buffer at pH 8.0.
z.
Pjlanzenphysiol. Ed. 102. S. 435-442. 1981.
440
JOHN KAMSTEEG
et al.
Table 2: Genetic control of NADPH: p-coumaroyl-CoA, 3-hydroxylase activity in petals of S. dioica plants. Genotype
Enzymatic activity':') with effector p-coumaric acid
Enzymatic activity':) with substrate p-coumaroyl-CoA
PIP
323
386
pip
141
none
,:.) The activity was determined in the Sephadex G-tSO fractions (M. W. 30,000) and is expressed as the oxygen consumption in the standard assay at 20°C in nMollmin/mg protein.
Kinetics Michaelis constants for the hydroxylMion reaction were determined by means of linear regression analysis on the Eadie-Hofstee linear transformation Cs/V versus Cs (Dowd and Riggs, 1965). The Km for p-coumaroyl-CoA was 0.2,uM. For the part in which a linear relationship between [FAD]/V and [FAD] was observed a Km was calculated of 1,uM for FAD. A Km of O.l,uM was found for NADPH. Maximal activity was obtained with substrate concentrations of about 10-4 M; at concentrations above 10- 3 M the reaction rate was inhibited. Other effectors or aromatic compounds at concentrations higher than 10- 3 M are non-competitive inhibitors. Also free Coenzyme A inhibited the reaction rate strongly.
Molecular weight determination The molecular weight of the enzyme was determined with a Sephadex G-150 column calibrated with cytOchrome c, chymotrypsinogen A, egg albumin, aldolase, and catalase as standards (Andrews, 1965). In the fractions corresponding with a molecular weight of 30,000, 60,000 and 120,000 dalton a maximum in hydroxylating activity was detected. The oxygen consumption in the 60,000 and 120,000 fraction was higher than in the 30,000 dalton fraction, but the oxygen consumption in these two fractions was only linear for a very short period. Dowex 1X2 treatment before Sephadex G-150 chromatography caused an increase of activity in the 30,000 dalton fraction. Without this step most of the activity was present in the 120,000 dalton fraction. It is remarkable that no peak in enzyme activity was detected which corresponded with a molecular weight of 90,000 daltons.
Spectrophotometric investigations The difference spectrum of the enzyme reduced with sodium dithionite in the presence and absence of carbon monoxide showed a photOdissociable peak at 450 nm, which is typical for cytochrome P-450 (Omura and SatO, 1964; Ambike et al., 1970). Additional evidence for the presence of cytochrome P-450 Z. PJlanzenphysiol. Bd. 102. S. 435-442. 1981.
p-Coumaroyl-CoA hydroxylase from Silene
441
was obtained from the appearance of an absorption peak at 421 nm in the difference spectra of the enzyme with either 1 mM p-chloromercuribenzoate or 4 Ofo sodium cholate. These reagents catalyze the conversion of cytochrome P-450 to cytochrome P-420 (Cooper et aI., 1964; Ichikawa and Yamaro, 1967). Discussion In this paper we describe an enzyme isolated from petals of PIP Silene dioica plants, which catalyzes the hydroxylation of p-coumaroyl-CoA to caffeoyl-CoA. This reaction is NADPH-dependent and uses molecular oxygen. The enzyme-bound FAD can be removed by Dowex 1X2 treatment or by precipitation with solid ammonium sulphate, but not by gel filtration. According to the criteria of Massey and Hemmerich (1975) this enzyme is a flavoprotein mono-oxygenase; NADPH is necessary for catalytic activity, the hydroxylation of an aromatic compound is catalyzed, and more important, p-coumaroyl-CoA is b(}th effector and substrate. In the presence of only NADPH there is no reduction of the enzyme-bound FAD. Addition of substrate p-coumaroyl-CoA causes an enormous stimulation of the NADPH oxidation. The effector role of the substrate can be simulated by substrate analogues which also increase the rate of reduction of the enzyme bound flavin by NADPH, but cannot serve as hydroxylatable substrates. The compounds, p-coumaric acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde, caused NADPH oxidation in our assay system. The reduction product of molecular oxygen in these cases is hydrogen peroxide rather than water, the product normally formed with hydroxylatable substrates. This could be demonstrated by the addition of catalase, which in the case of hydrogen peroxide formation, liberated again half the amount of molecular oxygen consumed. With the substrate p-coumaroyl-CoA no oxygen liberation could be detected after the addition of catalase to the assay. The compounds mentioned above therefore fulfill the conditions postulated by Massey and Hemmerich (1975) for non-substrate effectors. In plants homozygous recessive for gene P the p-coumaroyl-CoA hydroxylating activity is lost. So gene P is the structural gene for p-coumaroyl-CoA hydroxylation. Remarkably the oxidase activity is retained in homozygous recessive pip plants. With p-coumaric acid as non-substrate effecwr, hydrogen peroxide is still formed. Although there are strong indications that cytochrome P-450 is present in the purified Sephadex G-150 fractions which correspond with a molecular weight of 25-40,000 daltons, it is probably not involved in the hydroxylation mechanism. Poisening of cytochrome P-450 with carbon monoxide or other compounds had no influence on the reaction rate. We therefore suggest that the hydroxylation of p-coumaroyl-CoA occurs directly at the enzyme-bound FAD. Acknow ledgemen ts The authors are much indebted to Dr. R. Sutfeld, Botanisches Institut der Westfalischen Wilhelms-Universitat, Munster, Germany, for kindly supplying a sample of p-coumaroyl-
z. Pflanzenphysiol.
Bd. 102. S. 435-442. 1981.
442
JOHN KAMSTEEG et al.
CoA. We wish to thank Mr. George E. W. Thorig for his helpful remarks and Mr. D. Smit for preparing the illustration.
References AMBIKE, S. H., R. M. BAXTER, and N. D. ZAHID: The relationship of cytochrome P-450 levels and alkaloid synthesis in Claviceps purpurea. Phytochemistry 9, 1953-1958 (1970). ANDREWS, P.: The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606 (1965). DOWD, J. E. and D. S. RIGGS: A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations. J. Bioi. Chern. 240, 863-869 (1965). ENTSCH, B., D. P. BALLOU, and V. MASSEY: Flavin-oxygen derivatives involved in hydroxylation by p-hydroxybenzoate hydroxylase. J. BioI. Chern. 251, 2550-2563 (1976.) GRISEBACH, H.: Comparative biosynthetic pathways in higher plants. In: SWAIN, T., (Ed.): Chemistry in evolution and systematics pp. 487-511. Butterworths, London, 1973. HESS, D.: Der Einbau Methylgruppen-Markierter Ferulasaure und Sinapinsaure in die Anthocyane von Petunia hybrida. Planta 60, 568-581 (1964). - Die Wirkung von Zimtsauren auf die Anthocyansynthese in isolierter Petalen von Petunia hybrida. Z. Pflanzenphysiol. 56, 12-19 (1967). - Biochemische Genetik: eine Einfiihrung unter besonderer Beriicksichtigung hoherer Pflanzen. In: HESS, D., (Ed.): Phenole und Phenolderivate pp. 23-29. Springer-Verlag, Berlin, 1968. HOWELL, L. G., T. SPECTOR, and V. MASSEY: Purification and properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. J. Bioi. Chern. 247, 4340-4350 (1972). ICHIKAWA, T. and T. YAMANO: Reconversion of detergent- an{] sulfhydryl reagent-produced P-420 to P-450 by polyols and glutathione. Biochim. Biophys. Acta. 131,490-497 (1967). KAMSTEEG, J., J. VAN BREDERODE, and G. VAN NIGTEVECHT: Pleiotropic effect of a pelargonidin-hydroxylation gene in Silene dioica? Phytochemistry 15, 1917-1918 (1976). - - - Genetical and biochemical evidence that the hydroxylation pattern of the anthocyanin B-ring in Silene dioica is determined at p-coumaroyl-CoA stage. Phytochemistry 19,1459-1462 (1980). LOWRY, O. H., H. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MASSEY, V. and P. HEMMERICH: Flavin and Pteridine mono-oxygenases. In: BOYER, P. D. (Ed.): The enzymes Vol. XII. part B. Oxidation-rwuction, electron transfer (II), oxygenases and oxidases (I). pp. 191-253. Academic Press, New York, 1975. NIGTEVECHT, G. VAN: Genetic studies in dioecious Melandrium. I. Sexlinked and sex-influenced inheritance in Melandrium album and Melandrium dioicum. Genetica 37, 281306 (1966). OMURA, T. and R. SATO: The carbon monoxide-binding pigment of liver microsomes. J. Bioi. Chern. 239, 2370-2378 (1964). PARKES, G. D.: Mellor's modern inorganic chemistry. lIth w. pp. 397-399. Longmans, Green and Co. Ltd., London (1963). SPECTOR, T. and V. MASSEY: Studies on the effector specificity of p-hydroxybenzoate hydrocylase from Pseudomonas fluorescens. J. BioI. Chern. 247, 4679-4687 (1972).
Z. Pflanzenphysiol. Bd. 102. S. 435-442. 1981.