Characterization of cytochrome P450-dependent isoflavone hydroxylases from chickpea

Phytochemistry, Vol. 32, No. 3, pp. 653-657, 1993 printedin GreatBritain.

0031 9422/93$6.00+ 0.00 0 1993Pergamon PressLtd

CHARACTERIZATION OF CYTOCHROME P450-DEPENDENT ISOFLAVONE HYDROXYLASES FROM CHICKPEA STEPHAN CLEMENS, WALTER HINDERER, UTA WI~KAMPF

and WOLFGANG BARZ

Institutfiir Biochemie und Biotechnologie der PRanzen, WestfSlische Wilhelms-Universitlt,

Hindenburgplatz 55, W-4400 Miinster,

Germany (Received in revised form 1 September IN HONOUR

Key Word Index-Cicer induction; formononetin;

OF PROFESSOR

arietinum;

biochanin

MEINHART

1992)

ZENK’S

SIXTIETH

BIRTHDAY

Fabaceae; cytochrome P450; phytoalexin A; medicarpin; maackiain.

synthesis; elicitor

Abstract-Microsomal fractions from elicited chickpea cell cultures, roots and leaves in the presence of 0, and NADPH catalyse the 2’- and 3’-hydroxylations of the isoflavones formononetin and biochanin A. The enzymes were characterized as to their substrate specificity, cofactor requirements and inhibition by cytochrome P450 inhibitors. The activities, which are low or not measurable in non-elicited cultures, roots and leaves, are strongly induced upon biotic and abiotic elicitation. Their induction pattern, however, is different depending on the source of the microsbmal fraction. Induction of the 2’-hydroxylation of formononetin precedes accumulation of the chickpea phytoalexin medicarpin by two to four hours. The role of 2’-hydroxylation of formononetin in medicarpin synthesis is discussed.

INTRODUCTION

RESULTS

The phytoalexins of Cicer arietinum are the (-)-(6aR, 1laR)pterocarpans medicarpin and maackiain [ 11. Under stress conditions they are formed in all parts of the plant. In cell suspension cultures, phytoalexin formation can be elicited by treatment with fungal cell wall fragments [Z]. The biosynthesis of medicarpin and maackiain, first elucidated in red clover [3], proceeds via the constitutively formed isoflavone formononetin (Fig. 1) as a central intermediate. Hydroxylation in the 2’-position followed by a reduction reaction and ring closure to the pterocarpan structure yields medicarpin. In maackiain biosynthesis these two final steps are preceded by 3’hydroxylation, required for the formation of the methylenedioxy bridge and subsequent 6’-hydroxylation. Furthermore, in chickpea an analogous pathway also exists using the second constitutive isoflavone, biochanin A, in which all the aforementioned steps, with the exception of pterocarpan formation, occur leading to 2’-hydroxylated isoflavanones [4]. Therefore, four different isoflavone hydroxylations have to be anticipated in chickpea. Our work has been directed towards the identification, characterization and purification of the enzymes involved in phytoalexin biosynthesis in chickpea [4-6]. Here we present data on the isoflavone hydroxylases responsible for 2’- and 3’-hydroxylation of formononetin and biochanin A. The results show that these enzymes are cytochrome P450-dependent monooxygenases. A preliminary characterization and a report on the induction of these enzymes in cell suspension cultures of chickpea have already been published [7, 83.

Detection ofellzymatic activity Crude protein extracts or microsomal preparations of cultured chickpea cells treated with yeast cell wall elicitor were incubated with formononetin or biochanin A and various cofactors, e.g. NADH, NADPH, FAD, FMN, NAD+, NADP+. Isoflavones were extracted from the assay mixture with ethyl acetate and analysed by HPLC. After incubation of the microsomal fractions with NADPH and substrate the formation of new compounds could be detected. They were isolated by preparative HPLC and identified as 2’- and 3’-hydroxylated formononetin and biochanin A via GC-MS [7]. The respective enzyme activities were named formononetin 2’-hydroxylase (M’H), formononetin 3’-hydroxylase (F3’H), biochanin A 2’-hydroxylase (B2’H) and biochanin A 3’hydroxylase (B3’H), respectively. All four enzyme activities are summarized as isoflavone hydroxylases (IHDs). The standard enzyme assay was linear for 45 min and for protein concentrations of up to 200 pg ml-’ for all four reactions. Characterization

of the isojiavone hydroxylases

NADPH is essential for the 2’- and 3’-hydroxylations of formononetin and biochanin A. With NAD+, NADP+ or NADH alone no reaction could be measured. However, simultaneous addition of NADPH and NADH yields a synergistic effect. The activities are 34-47%

653

S. CLEMENSet al.

654

Fomwnonetin (R=H) Biochanin A (R=OH)

/

2’-OH-Fonnononetin (R=H) 2’-OH-Biochanin A (R=OH)

\

3’-OH-Fo mxmonetin (R=H) 3’-OH-Biochanin A @OH)

Fig. 1. Reactions catalyzed by isoflavone 2’- and 3’-hydroxylase. higher than with NADPH alone. With FAD and FMN no increase could be observed. The 2’- and 3’-hydroxylases display pH optima of 7.4 and 8.0, respectively. The temperature optimum is 30” for all four enzyme activities. An absolute requirement for oxygen was shown by removal of 0, from the assay by the glucose/glucose oxidase/catalase system. Under these conditions the hydroxylases are completely inactive. Substrate speciJicity

Various other compounds related to formononetin and biochanin A were incubated with microsomal preparations and NADPH under standard assay conditions. No reaction was detected with daidzein or genistein, the 4’OH precursors of formononetin and biochanin A, respectively, with formononetin-7-ethyl ether, 2’-hydroxyformononetin, vestitone and medicarpin. 5,7,4’-trimethoxyisoflavone and 6-hydroxyformononetin are transformed into unknown products. These results indicate a specificity of the IHDs For 4’-methoxyisoflavones and they also show that simultaneous dihydroxylations in positions 2’ and 5’ do not occur. The apparent K, values For F2’H and F3’H with formononetin as the substrate are 3.3 PM and 11.0 PM, and for B2’H and B3’H with biochanin A the values are 13.0 PM and 12.5 PM, respectively. Upon incubation of microsomal preparations from unelicited or elicited cells under standard conditions, hydroxylation of naringenin (5,7,4’-trihydroxyflavanone) to eriodictyol (5,7,3’,4’-tetrahydroxytlavonone) and of kaempferol (5,7,4’-trihydroxyflavonol) to quercetin (5,7,3’,4’-tetrahydroxyflavonol) could be observed (data not shown in detail). These reactions can be attributed to the presence of a flavonoid 3’-hydroxylase in these cells. These enzyme reactions are also known from soybean [9]. Furthermore, kaempferol and quercetin are constituents of chickpea [lo]. P450 dependence of the isojavone hydroxylases O2 and NADPH-requirement, the synergistic etfect of NADPH and NADH and the localization of the enzymes

in the microsomal Fraction suggested a cytochrome P450 dependence of the IHDs. Inhibitor studies were carried out to prove this hypothesis. The blue light-reversible inhibitory effect by CO is the best-established criterion For P450 involvement [ 11). Incubation of a microsomal preparation in a CO/O, (9:l) atmosphere in the dark under otherwise standard assay conditions led to an 88-96% inhibition of the four isoflavone hydroxylase activities while only a 19-22% inhibition was observed in a N,/O, (9:l) atmosphere in the dark. However, no reversion by blue light could be achieved. Several established P450-specific inhibitors were tested for their effect on the IHDs. I& values were determined graphically From dose-response curves. The results (Table 1) provide Further evidence For the cytochrome P450 character of the isoflavone hydroxylases. Cytochrome c and juglone are the most potent inhibitors. Noteworthy, in particular, is the differential inhibition of 2’- and 3’-hydroxylation by compounds such as BAS 110 and BAS 111. The different pH optima of 2’- and 3’-hydroxylation together with the inhibitor data suggest that at least two distinct proteins catalyse the Four reactions. The P450 content of microsomes from elicited chickpea cells was calculated from the CO difference spectrum (Fig 2) to 250 pm01 mg-’ protein, which is in the average

Table 1. Concentrations @cM) of cytcchrome P450 inhibitors which cause 50% inhibition of F2’H, F3’H, B2’H and B3’H activities Cytochrome P450 inhibitor Cytcchrome c Juglone Triadimefone Tetcyclacis Ketoconazol BAS 110 BAS 111 LAB 150978

F2’H

IC,, (PM) for

F3’H

B2’H

1

1

1

1 8

1

I

100 4 25 55 700 300

7 2 40 4 35 120

4 15 3 45 150

B3’H 1 1 130 18 140 40 250 500

Cytochrome P450-dependent isoflavone hydroxylases

0.04 0.03 0.02 % 0.01 -

rA \ + elicitor

O-O1 fi control

-0.01

L 8

400

I

I

425 450 475 Wavelength (nm)

500

Fig. 2 Absorption spectra of microsomal preparations from elicited (+ elicitor) and non-elicited (control) chickpea cells after chemical reduction and treatment ,with CO [17] depicting different cytochrome P456 content.

655

2.5 pkat kg- ’ protein (B2’H) were found (Fig. 3). Furthermore, F3’H activity is also not found in untreated roots and in contrast to the 2’-hydroxylases it is only very weakly induced. Phytoalexin accumulation in Mn2 +treated chickpea roots is clearly correlated with the expression of the different monooxygenase activities. Medicarpin (800 ,umol g- ’ fr. wt) and maackiain (120 pmolg-’ fr. wt) appear within 60 hr. Medicarpin accumulation follows F2’H induction with a time lag of about 2 hr (Fig. 4). In leaves the induction of F2’H following MnCl, treatment could also be demonstrated. After 36 hr an activity of 500 nkat kg- 1 protein was measured whereas in microsomal preparations from leaves of untreated plants no 2’-hydroxylation of formononetin could be detected. However, severe losses of P450-dependent enzyme activity occurred during the isolation of the microsomal fraction from leaves as documented by a comparison of accumulating phytoalexin amounts, protein concentrations and F2’H activities between roots and leaves of Mn2 +-stressed plants. Leaves accumulate twofold more phytoalexins than roots, contain less protein per gram fresh weight, but the specific activity measured for F2’H is less than 10% of the specific activity measured in roots. Similar losses were detected for CA4H.

range found in plant microsomes [ 121. Microsomes from non-elicited cells contain significantly less P450 protein (130 pm01 mg- 1 protein). Elicitor induction of the cinnamic acid 4-hydroxylase (CA4H) from 4 pkat kg- 1 protein to 18 pkat kg-’ protein accounts for part of the increase in P450 content. For the NADPH cytochrome P450 (c) reductase, the enzyme transferring electrons from NADPH to the P450 protein, an activity of about 2 mkat kg-’ protein was measured both in non-elicited and elicited cells. Induction of the isoflavone hydroxylases

In non-elicited chickpea cells only very low isoflavone hydroxylase activities of 0.3-1.5 pkat kg- 1 protein can be measured. Upon treatment of 72 hr-old cultures with a yeast cell wall elicitor, all four IHDs are strongly induced reaching maxima of 12 pkatkg-’ protein (F2’H), 15 pkat kg- ’ protein (F3’H), 6.5 @at kg- ’ protein (B2’H) and 14.5 pkat kg- ’ protein (B3’H) after 8 hr and these activities rapidly decline thereafter. Phytoalexin accumulation reaches a maximum 12 hr after elicitation. In roots and leaves of chickpea the synthesis of medicarpin and maackiain is readily elicited by addition of MnCl, to the culture fluid (Clemens and Barz, unpublished results). Thus, the induction of the monooxygenases in plant tissue can easily be studied. The induction pattern of the IHDs found in 6-&day-old roots is different from the situation observed in cell suspension cultures. 3’-Hydroxylase activity of biochanin A was expressed in untreated roots (2.2 &at kg- ’ protein), and it did not increase under Mn2+ stress. On the other hand the constitutively not detectable F2’H and B2’H activities are strongly induced. Twelve hours after MnCl, addition, maximum activities of 5.5 @at kg-’ protein (F2’H) and

4

8

12

16

20

24

Xme after elicitation

36 (hr)

Fig. 3. Time course of the induction of biochamin A 3’-hydroxylase (O-O), biochanin A 2’-hydroxylase (A -A) and formononetin 2’-hydroxylase (0-D) in chickpea roots stressed with MnCl, Formononetin 3’-hydroxylase failed to be induced to any significant extent.

.!j

11000

6r

4 8 12 16 20 24 lime after elicitation (hr) Fig. 4. Correlation of formononetin 2’-hydroxylase (0-O) induction and medicarpin accumulation (bars) in Mns+-treated chickpea roots.

S. CLEMENS et al.

656 DISCUSSION

The four isoflavone hydroxylase activities measurable in microsomal fractions from chickpea cell cultures, roots and leaves require 0, and NADPH and are strongly inhibited by CO, cytochrome c and various P4SO-specific inhibitors. Although no blue light reversion of CO inhibition could be achieved, these data clearly show that the 2’and 3’-hydroxylations of formononetin and biochanin A belong to the rapidly increasing number of reactions identified in plants as cytochrome P450-dependent [ 131. In chickpea cell cultures the IHDs are strongly induced upon treatment with a yeast elicitor. For the F2’H this was confirmed in cell cultures of alfalfa [14]. In roots mainly F2’H and B2’H are induced by MnCl, addition to the culture fluid, while B3’H is constitutively expressed and F3’H is only weakly induced. The metabolic role of B3’H in roots is readily explained by the constitutive accumulation of the isoflavone pratensein (i.e. 3’hydroxybiochanin A) in this organ of chickpea [4]. Induction of F2’H in cell cultures and roots (Fig. 4) precedes the accumulation of medicarpin by 2 hr which again supports our previous observations [8]. This correlation and the fact that the other enzymes involved in the transformation of formononetin into medicarpin (i.e. isoflavone reductase and pterocarpan synthase) show significant activities in non-elicited cells lead us to suggest that the FZ’H is a site of regulatory action. Accumulation of phytoalexins in chickpea plants can be elicited by addition of MnCl, to the culture fluid. From leaves of MnCl,-treated plants we were able to prepare microsomal fractions with detectable cytochrome P450-dependent activities. However, severe losses of activity occurred probably due to the high content of phenolic material, lipases and proteases [ 123. 2’- and 3’-hydroxylations differ in pH optima and sensitivity towards inhibitors like BAS 110 and BAS 111 (Table 1). Furthermore, F3’H and B3’H activities in roots exhibit a pronounced difference in their constitutive expression. Since F3’H and B3’H show similar K, values for their respective substrates, these findings suggest that at least three different proteins account for the four measurable isoflavone hydroxylase activities in chickpea. The question as to whether one or two enzymes catalyse 2’-hydroxylation of formononetin and biochanin A cannot be decided on the basis of the presented data but requires successful purification and reconstitution of the 2’-hydroxylase activities.

EXPERIMENTAL

Cell cultures. All cell cultures were established in our laboratory and grown as previously described [15]. The cell line derived from cultivar ILC 3279 was used for the enzymic investigations. The cultures were elicited by addition of 40 mg yeast extract/40 ml medium. Plant material. Chickpea seeds were soaked for 18 hr, germinated at 20” in the dark for 48 hr and grown under a 14 hr light period. Six- to eight-day-old seedlings or 2-3-

week-old plants were elicited by addition of 10mM MnCl, to the culture fluid. Preparation of microsomes. (a) From cell cultures: microsomal preparations from cell cultures were obtained as previously described [8]. (b) From roots: roots were harvested at various times after M&l, treatment and homogenized in a mortar in degassed 0.08 M Tris-HCl (pH 7.5) containing 0.4 M sucrose, 10 mM 2mercaptoethanol, 40 mM ascorbic acid, 0.1% (w/v) BSA and 0.3 gg- ’ fr. wt. Dowex 1 x 2 (buffer A). The homogenate was filtered through cheesecloth and centrifugedfor 10 min at 10000 g. The supernatant was centrifuged for 1 hr at 100000 g. The resulting microsomal pellets were resuspended in 0.05 M K-Pi (pH 7.5) containing 0.4 M sucrose and 2 mM DTE (buffer B) and homogenized with a Potter-Elvehjem homogenizer. (c) From leaves: leaves from 2-3-week-old chickpea plants were harvested at various times after MnCl, treatment and homogenized with a Waring blender (3 set low-8 set high) in buffer A. The homogenate was filtered through cheesecloth and centrifuged for 20 min at 24000 g. To the resulting supernatant 1 M MgCl, was added to a final concentration of 30 mM. The suspension was gently stirred for 20 min and centrifuged for 20 min at 40000 g. Resuspension and homogenization of the microsomal pellets were performed as described for root material. Microsomal preparations from cell cultures, roots and leaves could be frozen in liquid N, and stored at - 70” for several weeks without loss of activity. Enzyme assays. The standard assay for IHD activities contained in a final volume of 500 ~1, 50-100 fig microsomal protein, 0.05 M K-Pi (pH 7.5), 0.4 M sucrose, 2 mM DTE, 1 mM NADPH, 1OOpM formononetin or biochanin A. After 45 min incubation at 30” the reactions were stopped and the assay mixture extracted by addition of 800 ~1 EtOAc. The organic phase was evapd to dryness and the extracted compounds redissolved in MeOH and analysed by HPLC. HPLC analysis was carried out using an RP-18 column (125 x 4 mm, 7 q). The solvent system was acetonitrile azeotrope/0.15% phosphoric acid. Substrates and products were sepd by an acetonitrile azeotrope gradient from 40% to 70% in 18 min. Formononetin and its derivatives were detected at 248 nm, biochanin A and its derivatives were detected at 261 nm with a photodiode array detector. All other compounds were measured at their maximum absorption. NADPH-cytochrome P450 (c) reductase was measured according to ref. [16]. Cytochrome P450 content was measured as described by Omura and Sato [ 171 assuming an absorption roe& cient of 91 mM_’ for the 448-490 nm absorption difference. Microsomal protein was estimated by the method of Bradford [18] using BSA as a standard. Acknowledgements-Financial

support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. Dr G. Retzlaff (BASF, Limburgerhof, F.R.G.) kindly donated inhibitor compounds.

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