- Email: [email protected]
[29] Cinnamic acid hydroxylase activity in plant microsomes
[291
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solution containing 0.5 M NaCl (buffer B). The AOS activity from flaxseed eluted at ~20 min (Fig. 5A).
Chromatofocusing FPLC (Mono-P Column) Finally, following buffer exchange using a gel filtration (PD-10 column, Pharmacia), the enzyme was purified on a Mono-P H R 5/20 chromatofocusing column (Pharmacia) (Fig. 5B and Table I). The two isozymes differ in a single amino acid at the N terminus, and the major isozyme was cloned and sequenced2 The isozymes have indistinguishable UV-VIS spectra, typical of a P450 in the high-spin ferric state, al Each catalyzes allene oxide synthesis with a turnover number of ~>1000 secq. u
Emulgen Detergent Removal For microsequencing, removal of Emulgen 911 is desirable. It cannot be removed effectively by dialysis or with Centricon spin concentrators. Injection of the sample on a Mono-Q column, followed by extensive washing with detergent-free buffer, and then elution with a salt gradient including octylglucoside (20 mM) is effective. Octylglucoside can be dialyzed. The denatured enzyme can also be resolved from Emulgen 911 on RP-HPLC (Vydac C4 column and a water/acetonitrile gradient), although this is not effective on a preparative scale. Acknowledgment This work was supported by N I H Grant GM-49502.
[29] C i n n a m i c A c i d H y d r o x y l a s e A c t i v i t y in Plant Microsomes
By
FRANCIS D U R S T , IRI~NE BENVENISTE, M I C H E L SCHALK, a n d DANII~LE W E R C K - R E I C H H A R T
Introduction Cytochrome P450 (P450) enzymes were characterized in plant tissues in the early 1970s. For almost two decades these enzymes attracted little attention and progress was slow. The two main reasons for this were (1) technical difficulties in dealing with poorly abundant membrane proteins METHODS IN ENZYMOLOGY,VOL. 272
Copyright © 1996 by AcademicPress, Inc. All rightsof reproductionin any formreserved.
260
PLANTP450S
NH 3 Phenylalanine _ ~ ~ PAL
C02H CA4H
trans -cinnamic acid
~ HO-
[29]
~tCO2H "~
para-coumaric acid
Lignin Suberin .~ Phytoalexins ~_ Pigments Aroma ~_ UV protectanls
FIG. 1. The two first reactions in phenylpropanoid synthesis. PAL, phenylalanine ammonia lyase.
in tissues harboring numerous and active proteases and lipases, the presence of hydrophobic and extremely "sticky" pigments like chlorophyll, and a vacuole filled with inhibitory secondary metabolites that the plant cell produces but cannot excrete, and (2) the assumption that plant P450, being so similar to mammalian hepatic enzymes, was bound to catalyze the same reactions. In recent years it was realized that plant P450s catalyze unique reactions 1-3 on substrates that have no equivalent in animals. Since then, progress has been so rapid that at present the plant P450 families span from CYP71 to CYP96 and probably many more are to come. The cinnamic acid 4-hydroxylase (CA4H) is the earliest and best studied P450 activity in plants. The discovery that CA4H activity was microsomal and required NADPH 4 was followed by the demonstration of the P450 nature of the enzyme.5'6The enzyme was first purified,7 cloned,s and heterologously expressed 9 in our laboratory. CA4H catalyzes the conversion of trans-cinnamic acid to trans-4hydroxycinnamic acid (para-coumaric acid), committing the C6-C3 carbon skeleton from phenylalanine toward the phenylpropanoid pathway (Fig. 1). This is the second reaction, and the first oxidative step, in the general phenylpropanoid pathway, which is common to all plants. Phenylpropanoids, and their derivatives, constitute an extremely diversified family of molecules with important biological functions or activities: precursors for 1F. Durst, in "Frontiers in Biotransformation" (K. Ruckpaul and H. Rein, eds.), Vol. 4, p. 191. Akademie-Verlag, Berlin, 1991. 2 F. Durst and I. Benveniste, Handb. Exp. Pharmacol. 105, 293 (1993). 3 p. Bolwell, K. Bozak, and A. Zimmerlin, Phytochemistry 37, 1491 (1994). 4 D. W. Russell, J. Biol. Chem. 246, 3870 (1971). 5 I. Benveniste and F. Durst, C. R. Hebd. Seances Acad. Sci., Ser. D 278, 1487 (1974). 6 M. Potts, R. Weklych, and E. E. Conn, J. Biol. Chem. 249, 5019 (1974). 7 B. Gabriac, D. Werck-Reichhart, H. G. Teutsch, and F. Durst, Arch. Biochem. Biophys. 288, 302 (1991). 8 H. G. Teutsch, M.-P. Hasenfratz, A. Lesot, C. Stoltz, J.-M. Gamier, J.-M. Jeltsch, F. Durst, and D. Werck-Reichhart, Proc. Natl. Acad. Sci. U.S.A. 90, 4102 (1993). 9 p. Urban, D. Werck-Reichhart, H. G. Teutsch, F. Durst, S. Regnier, M. Kazmaier, and D. Pompon, Eur. J. Biochem. 222, 843 (1994).
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lignin and suberin, pigments, aroma, defense molecules (phytoalexins), antioxidants, and U V protectants. Many of these compounds are specific to certain plant families or species, and a number of other P450 enzymes are involved in these reactions. 1-3 The major difference between C A 4 H and most other plant P450s which appear to be involved in species specific reactions is that C A 4 H is probably present in all plants and in almost all tissues. Assaying CA4H is therefore a foolproof way to ascertain that microsomal preparations contain intact and active P450 electron transfer chains, and provides a sort of internal standard when extraction conditions are modified. This chapter covers the following topics: (i) the nature of the enzyme source (plant species and type of tissue), (ii) induction of CA4H activity, (iii) preparation of microsomes, and (iv) two reliable and simple assays. N a t u r e of Plant a n d T i s s u e Preparing microsomes from adult green tissues should be avoided when possible. At this stage, cells contain an enormous vacuole, and high mechanical forces are required to break the rigid walls. This leads to intensive mixing of membrane fractions with pigments and phenolics: activities are weak and difference spectra are marred by a high absorbance background and spurious redox shifts. It is better to use young (2-8 days) etiolated seedlings, root or tuber slices, and cell suspension cultures. Cell cultures are beyond the scope of this chapter. We will briefly describe a standard procedure to grow Vicia seedlings and prepare Jerusalem artichoke tuber slices. Seedlings Vicia sativa seeds are immersed for 10 min in a 0.3% sodium hypochlorite solution, washed with running tap water and distilled water, and soaked for 6 hr in distilled water at 27 °. Seeds are then sown on six layers of watersoaked filter paper placed in covered 20 x 20-cm plastic boxes and incubated for 4 days at 26 ° in total darkness. Seedlings may be used as such or further induced. In any case, the seed teguments that are rich in tannins and other polyphenols are removed manually prior to microsome preparation. Tuber Slices Reproduction of Helianthus tuberosus is vegetative. We use a clone from variety 'blanc commun' isolated in 1948, which ensures great genetic homogeneity. Other varieties purchased from stores or markets, however, show similar activities. Tubers are harvested in the fall and stored in open
262
PLANT P450S
[29]
polyethylene bags in darkness at 4 °. In the case of storage tissues like tubers, C A 4 H is very low in intact tissue and is strongly enhanced by slicing, i.e., wounding, and by chemical treatments (see below). After harvest, a 3-week vernalization period is required before consistent and reproducible results are obtained.
I n d u c t i o n of CA4H C A 4 H is a highly inducible enzyme, 1,3 and since the reaction product, p-coumarate, is a precursor to so many different compounds and involved in different processes, the enzyme is induced by a wide range of factors. This may be of great help in characterizing the activity in rare or difficult to sample tissues. On the other hand, it implies that strict standardization of growth and handling conditions is necessary to achieve reproducibility.
Wounding, Infection, and Light Any kind of mechanical wounding or exposure to fungi, microbes, or viruses elicits the production of phytoalexins and tissue repair mechanisms. C A 4 H is involved in both. The elicitation of C A 4 H with fungal extracts will not be treated here, but it should be noted that an unnoticed infection may cause important activity variations. Wound Induction of H. tuberosus Tuber. Tubers are peeled, washed with distilled water, and cut in 1-mm-thick slices. Slices are washed for 2 hr in running water, rinsed with distilled water, and aged in a 2-liter Erlenmeyer flask containing 1.5 liter distilled water for 50-120 g tissue. The slices are aerated by a 4-liter min -1 stream of hydrated and filtered air, and aged in the dark at 25-30 °. It is important to consider that induction is a rapid and transient phenomenon, which reaches a peak after 18-24 hr and declines thereafter. 1° Long storage of the tubers at 4 ° before use (i.e., release of dormancy) delays and lowers maximum activity. Timing of tissue sampling for microsome preparation is thus essential for adequate activity and reproducibility. Light Is a Known Inducer of CA4H Activity. This effect is mediated by the phytochrome photoreceptor, u which may be activated either by continuous irradiation with low intensity far-red (730 nm) light or pulses of red (660 nm) light. U n d e r both conditions, C A 4 H is considerably enhanced, while chlorophyll synthesis remains minimal. As with other induction systems, it is essential to establish a time course of induction first, so sampling a0I. Benveniste, J.-P. Salaiin, and F. Durst, Phytochemistry16, 69 (1977). aa I. Benveniste, J.-P. Sala0n, and F. Durst, Phytochemistry17, 359 (1978).
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of the seedlings is made at the plateau for activity measurements. Red light induces C A 4 H in the seedlings of many plant species, but does not increase the enzyme activity in tuber slices. Seedlings are first grown for 4-7 days in the dark before irradiation. For irradiation, a projector equipped with a 660-nm interference filter is automatically switched on for 2 min every 6 hr. Even a single 5-min red pulse followed by 18 hr darkness before preparation of microsomes may double C A 4 H activity, n Chemicals
Early work from our laboratory had demonstrated that C A 4 H activity was increased when tuber slices or seedlings were exposed to certain chemicals like 2,4-dichlorophenoxyacetic acid, 12 several herbicides, phenobarbital, ethanol or to manganese ions, a3 and by aminopyrine. TM The response of C A 4 H to these compounds varies considerably depending on plant species and tissue. For example, MnC12, which increases strongly the activity in tuber slices, 13 had deleterious effects on pea seedlings. We will describe the induction of C A 4 H in H. tuberosus slices. The H. tuberosus tuber is sliced, washed, and aged in aerated water in the dark as described earlier for wound induction. Chemicals are dissolved directly into the aging water or, if needed, in a small volume of dimethyl sulfoxide added to the aging medium. The most effective inducers are 25 m M MnCI2, 20 m M aminopyrine, and 4 m M phenobarbital. The p H of the MnCI2 solution is adjusted to 7 with NaOH. As noted previously, time course studies are required to determine the peak of C A 4 H activity. For the previously mentioned inducers, maxima are observed at 48 to 72 hr after slicing. Chemical induction of 3- to 5-day-old seedlings can be achieved in a similar way. After removal of the seed tegument, seedlings are aged in an aqueous solution as described for H. tuberosus slices.
P r e p a r a t i o n of Microsomes Standard Extraction Buffer for Tuber Slices
Just before extraction, add 100 m M sodium phosphate buffer, p H 7.4, containing I m M E D T A and 250 m M sucrose; 15 m M 2-mercaptoethanol, 40 m M sodium ascorbate. Check p H after adding sodium ascorbate. 12p. Adel6, D. Reichhart, J.-P. Salatin,I. Benveniste,and F. Durst, PlantSci. Lett. 22, 39 (1981). 13D. Reichhart, J.-P. Salaiin, I. Benveniste, and F. Durst, Plant Physiol. 66, 600 (1980). 14R. Fonn6-Pfister, A. Simon, J. P. Salaiin, and F. Durst, Plant Sci. 55, 9 (1988).
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PLANT P450s
[29]
Standard Extraction Buffer for V. sativa Seedlings Just before extraction, add 100 m M sodium phosphate buffer, p H 7.4, containing 1 m M E D T A and 10% glycerol (v/v); 15 m M 2-mercaptoethanol, 40 m M sodium ascorbate, 10% (w/w of fresh tissue) insoluble PVP, and 1 m M phenylmethylsulfonyl fluoride (from a stock solution in methanol).
Additions Modifications of the extraction buffer may improve enzyme recovery, depending on plant materials. If the extraction medium turns brown after grinding with tissues and 2-mercaptoethanol no longer "smells," increase concentration or add dithiothreitol or glutathione. Significant improvement can be achieved by adding: 1% (w/v) bovine serum albumin 0.6% (w/v) Amberlite XAD-4, 10 m M NaS2Os, and 1% (w/v) insoluble PVP as described for the preparation of enzymes involved in terpene synthesis as 0.5% Dowex 1 × 2 (w/fresh weight) or pulverulent active charcoal (1-3% w/fresh weight) Dowex and charcoal are very effective in removing inhibitory polyphenols but they also adsorb protein! The effects of several of these and other additives have been recently compared. 16
Grinding and Centrifugation All operations are performed on ice. Tuber slices or Vicia seedlings are homogenized in 1.5 volume chilled extraction buffer with a Warring blender or with an Ultra-Turrax at 8000 rpm. Homogenates are filtered through a 50-/zm nylon mesh and centrifuged for 15 min at 10,000 g. The supernatant is then centrifuged for 60 min at 100,000 g. Resuspending the microsome pellet in 100 m M sodium pyrophosphate buffer containing 10 m M 2-mercaptoethanol and running a second centrifugation may improve specific activity, but it decreases C A 4 H recovery. If an ultracentrifuge is not available or if large volumes are to be handled, add MgC12 (from a 1 M stock solution) to the 10,000-g supernatant to a final concentration of 30 mM, stir for 20 min, and centrifuge for 20 min at 40,000 g. The yield is lower, but specific activities are sometimes higher using this method. 15I. M. Rajaonariviny,J. Gershenzon,and R. Croteau, Arch. Biochem. Biophys. 296,49 (1992). 16A. E. Haack and N. E. Balke, Pestic. Biochem. Physiol. 50, 92 (1994).
[29]
CINNAMIC ACID HYDROXYLASE
265
The 100,000-g pellet (or the MgC12 precipitation pellet) is resuspended in sodium phosphate, p H 7.4, containing 1.5 m M 2-mercaptoethanol and 30% (v/v) glycerol to a final concentration of 5-7 mg microsomal protein m1-1. If stored at - 2 0 °, activity is stable for at least 2 months. Two CA4H Assays The two methods routinely used in our laboratory are described. H P L C determination has also been used successfully, but is more time-consuming. 9,17
Using Radiolabeled Cinnamic Acid The use of [3-14C]trans-cinnamic acid (Isotopchim, B.P. 16, F-04310 Ganobie, France) provides an easy and sensitive assay for CA4H. A 4-/.~Ci m1-1 [3-14C]cinnamate stock solution is prepared in 100 m M sodium phosphate, p H 7.4, and is stored as 2-ml aliquots at - 2 0 °. The exact cinnamate concentration of this stock solution is determined from its absorbance at 268 nm using the eM: 20,400. Depending on the C A 4 H activity in the microsomes to be studied, different amounts of cold substrate are added to this radiolabeled stock. A final cinnamate concentration ranging from 75 to 200/~M is often needed to ensure reaction linearity over 10-20 min with microsomes from induced plant tissues. Time (2 to 30 min) and protein concentration (0.05 to 0.5 mg in 200 /zl) are adjusted such that less than 50% of substrate is converted, ensuring saturation of the enzyme: the Km for cinnamate is in the 1 - 8 / ~ M range, depending on enzyme source. A control reaction, with HC1 added before N A D P H , is run to obtain the background and to make sure no contaminant running with an R~ comparable to that of coumarate is present in the substrate (it may happen occasionally). A typical assay with induced H. tuberosus microsomes contains 175 ~ M trans-cinnamic acid 0.5 m M N A D P H 0.1 mg microsomal protein 200 ~1 final sodium phosphate buffer 0.1 M, pH 7.4 When assays are incubated longer than 10 min, an N A D P H regeneration system (10 m M glucose 6-phosphate and 0.5 units glucose-6-phosphate dehydrogenase) is added to the incubation mixture. As the addition of N A D P H to plant microsomes usually results in rapid P450 inactivation, the reaction is started by the addition of N A D P H . After a 10-min incubation at 27 °, the reaction is stopped with 20 ~1 4 N HCI, and 17C. B. Stewart and M. A. Schuler, Plant Physiol. 90, 534 (1989).
266
PLANTP450s
[29]
10 /xl of a mixture of nonlabeled cinnamic and coumaric acids (100/xg each) is added. A 100-/xl aliquot of reaction medium is spotted onto a precoated TLC silica gel plate 60 F254 (Merck) and is developed in the organic phase from a toluene/acetic acid/water (6/7/3) mixture. Rf is 0.54 for cinnamate and 0.3 for para-coumarate. Activity is directly determined with a thin layer analyzer (Berthold LB 2820-1). Alternatively, para-coumarate and cinnamate are visualized as purplish-blue fluorescent spots under 254-nm UV light, and silica is scraped and counted in a 5-ml Ready organic scintillation cocktail (Beckman). The reaction is usually linear for at least 10 min, but the enzyme is inactivated at incubation times over 20 rain at 27° or 10 min at 30°. The optimal pH for CA4H is around 7.4, with half-maximal activities at pH 6.2 or 8. A significant activity increase can be achieved by increasing the ionic strength of the incubation buffer (i.e., using 100 mM rather than 25 mM sodium phosphate or 100 mM Tris buffer). For the yeast-expressed enzyme, inhibition was observed when the CA4H concentration was increased above 6 pmol in the assay, is
Fluorescent Determination of 2-Naphthoate Hydroxylation Radioactivity measurements are not always possible or desired. An alternative, stemming from our recent studies of the substrate specificity of yeast expressed CA4H, TM is a very fast and sensitive fluorimetric method, a9 We have demonstrated that 2-naphthoic acid perfectly mimics transcinnamate in the CA4H active site and is metabolized with an efficiency (Km and Vmax)comparable to that of cinnamate. The product of the reaction, 6-hydroxy-2-naphthoate, is strongly fluorescent. This property can be used for a rapid but reliable assay of the CA4H activity. The optimal excitation wavelength of 6-hydroxy-2-naphthoate varies slightly with the composition of the buffer and with the concentration of NADPH and substrate in the assay. In 100 mM sodium phosphate, pH 7.4, and in the presence of 50/xM NADPH, its maximum excitation is 299 nm, with an emission peak at 443 nm. The substrate does not interfere with the readings, and a direct and continuous measurement of fluorescence in the incubation medium is possible. However, this assay cannot be used with the microsomes contaminated with pigments (e.g., chlorophyll). A typical fluorescent CA4H assay contains 100/~M 2-naphthoic acid (Sigma) 18 M. Pierrel, Y. Batard, M. Kazmaier, C. Mignotte-Vieux, F. Durst, and D. Werck-Reichhart, Eur. J. Biochem. 224, 835 (1994). 19 M. Schalk, Y. Batard, A. Seyer, A. Zimmerlin, F. Durst, and D. Werck-Reichhart, to be published.
[29]
CINNAMICACID HYDROXYLASE
267
50 t i M N A D P H 1 m M glucose 6-phosphate 0.5 units glucose-6-phosphate dehydrogenase 0.1-0.5 mg microsomal protein 2 ml final sodium phosphate 100 mM, p H 7.4 The fluorescence of the reaction mixture without N A D P H is recorded for approximately 3 min to allow temperature equilibration (30 °) and to check for the activity in the absence of N A D P H (this step can be shortened by using a preheated buffer). N A D P H is then added to the reaction medium, and the increase in fluorescence (excitation: 299 nm; emission: 443 nm) is recorded for an additional 2 to 5 min. The reaction is usually linear for at least 10 min. Calibration of the assay is p e r f o r m e d using known amounts of 6-hydroxy-2-naphthoate (Lancaster Synthesis, Morecambe, UK). Some Typical CA4H Activities Enzyme source H. tuberosus
tuber
Wheat seedlings Vicia seedlings
Treatment Dormant Slicing + 24 hr aging Slicing + 56 hr aging in 25 mM MnC12 5 days old 4 days old 4 days old + 48 hr aging in aminopyrine
Activity (pmol sec i rag-l) 0-1 40 200 2 1 6
W h e n All A t t e m p t s H a v e Failed As already discussed, with some plants it might be impossible or very difficult to obtain measurable and reproducible C A 4 H activity. It is our experience that C A 4 H protein or transcript levels measured by Western or Northern blotting do provide a reasonable estimate of C A 4 H activity even when working with green tissues from nonlaboratory plants, i.e., plants collected in the natural environment. 2° However, the use of antibodies requires the preliminary purification of a small amount of h o m o g e n e o u s enzyme, not an easy task, although several purification methods have been published. 7,21,22 It is further hampered, at least for use on Western blots, by their p o o r cross-reactivity when the boundaries of plant families are 20y. Batard, Ph.D. Thesis, Universit6 Louis Pasteur, Strasbourg, (1995). 21M. Mizutani, D. Ohta, and R. Sato, Plant Cell PhysioL 34, 481 (1993). 22D. Werck-Reichhart, I. Benveniste, H. Teutsch, F. Durst, and B. Gabriac, AnaL Biochem. 197, 125 (1991).
268
PLANTP450S
[301
crossed. 23 This makes cDNA probes particularly attractive. CA4H has now been cloned from a range of species: H. tuberosus (Z17369), Phaseolus aureus (L07634), Medicago sativa (Ll1046), Catharanthus roseus (X69788, Z32563), A rab idopsis thaliana (T04086), Pisum sativum (U29243), and Zinnia elegans (U19922). These may be obtained from the authors or recloned using PCR or RT-PCR methods. CA4H is well conserved (generally over 80% positional nucleotide identity between different species) and crosshybridization on Northern blot seems more extended than with Western blotting. 23 D. Werck-Reichhart, Y. Batard, G. Kochs, A. Lesot, and F. Durst, Plant Physiol. 102, 1291 (1993).
[30]
Isolation of Plant and Recombinant B y B A R B A R A A N N HALK1ER, O L E SIBBESEN,
CYP79 and
B I R G E R LINDBERG M t 0 L L E R
Introduction We have developed an isolation procedure for CYP79, also called P450tyr, which is a key enzyme in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. x Cyanogenic glucosides are secondary plant products which, on hydrolysis, release HCN. CYP79 is a multifunctional N-hydroxylase converting tyrosine into p-hydroxyphenylacetaldoxime by two consecutive N-hydroxylations, of which the first produces N-hydroxytyrosine and the second N,N-dihydroxytyrosine. 2 The latter dehydrates to 2-nitroso-3-(p-hydroxyphenyl)propionic acid which decarboxylates into the oxime. The multifunctional property of CYP79 has been confirmed by reconstitution experiments using recombinant CYP79 expressed at high levels in Escherichia coli. 3 This chapter focuses on the generally applicable procedures developed for the isolation of plant and recombinant CYP79. In addition, a strategy is suggested for successful expression of microsomal P450s in E. coli. 1 0 . Sibbesen, B. Koch, B. A. Halkier, and B. L. MOiler, Proc. Natl. Acad. Sci. U.S.A. 91, 9740 (1994). 2 0 . Sibbesen, B. Koch, B. A. Halkier, and B. L. M¢ller, J. Biol. Chem. 270, 3506 (1995). 3 B. A. Halkier, H. L. Nielsen, B. Koch, and B. L. Mealier, Arch. Biochem. Biophys. 322, 369 (1995).
METHODS IN ENZYMOLOGY,VOL. 272
Copyright © 1996by AcademicPress, Inc, All rightsof reproductionin any form reserved.
CINNAMIC ACID HYDROXYLASE
259
solution containing 0.5 M NaCl (buffer B). The AOS activity from flaxseed eluted at ~20 min (Fig. 5A).
Chromatofocusing FPLC (Mono-P Column) Finally, following buffer exchange using a gel filtration (PD-10 column, Pharmacia), the enzyme was purified on a Mono-P H R 5/20 chromatofocusing column (Pharmacia) (Fig. 5B and Table I). The two isozymes differ in a single amino acid at the N terminus, and the major isozyme was cloned and sequenced2 The isozymes have indistinguishable UV-VIS spectra, typical of a P450 in the high-spin ferric state, al Each catalyzes allene oxide synthesis with a turnover number of ~>1000 secq. u
Emulgen Detergent Removal For microsequencing, removal of Emulgen 911 is desirable. It cannot be removed effectively by dialysis or with Centricon spin concentrators. Injection of the sample on a Mono-Q column, followed by extensive washing with detergent-free buffer, and then elution with a salt gradient including octylglucoside (20 mM) is effective. Octylglucoside can be dialyzed. The denatured enzyme can also be resolved from Emulgen 911 on RP-HPLC (Vydac C4 column and a water/acetonitrile gradient), although this is not effective on a preparative scale. Acknowledgment This work was supported by N I H Grant GM-49502.
[29] C i n n a m i c A c i d H y d r o x y l a s e A c t i v i t y in Plant Microsomes
By
FRANCIS D U R S T , IRI~NE BENVENISTE, M I C H E L SCHALK, a n d DANII~LE W E R C K - R E I C H H A R T
Introduction Cytochrome P450 (P450) enzymes were characterized in plant tissues in the early 1970s. For almost two decades these enzymes attracted little attention and progress was slow. The two main reasons for this were (1) technical difficulties in dealing with poorly abundant membrane proteins METHODS IN ENZYMOLOGY,VOL. 272
Copyright © 1996 by AcademicPress, Inc. All rightsof reproductionin any formreserved.
260
PLANTP450S
NH 3 Phenylalanine _ ~ ~ PAL
C02H CA4H
trans -cinnamic acid
~ HO-
[29]
~tCO2H "~
para-coumaric acid
Lignin Suberin .~ Phytoalexins ~_ Pigments Aroma ~_ UV protectanls
FIG. 1. The two first reactions in phenylpropanoid synthesis. PAL, phenylalanine ammonia lyase.
in tissues harboring numerous and active proteases and lipases, the presence of hydrophobic and extremely "sticky" pigments like chlorophyll, and a vacuole filled with inhibitory secondary metabolites that the plant cell produces but cannot excrete, and (2) the assumption that plant P450, being so similar to mammalian hepatic enzymes, was bound to catalyze the same reactions. In recent years it was realized that plant P450s catalyze unique reactions 1-3 on substrates that have no equivalent in animals. Since then, progress has been so rapid that at present the plant P450 families span from CYP71 to CYP96 and probably many more are to come. The cinnamic acid 4-hydroxylase (CA4H) is the earliest and best studied P450 activity in plants. The discovery that CA4H activity was microsomal and required NADPH 4 was followed by the demonstration of the P450 nature of the enzyme.5'6The enzyme was first purified,7 cloned,s and heterologously expressed 9 in our laboratory. CA4H catalyzes the conversion of trans-cinnamic acid to trans-4hydroxycinnamic acid (para-coumaric acid), committing the C6-C3 carbon skeleton from phenylalanine toward the phenylpropanoid pathway (Fig. 1). This is the second reaction, and the first oxidative step, in the general phenylpropanoid pathway, which is common to all plants. Phenylpropanoids, and their derivatives, constitute an extremely diversified family of molecules with important biological functions or activities: precursors for 1F. Durst, in "Frontiers in Biotransformation" (K. Ruckpaul and H. Rein, eds.), Vol. 4, p. 191. Akademie-Verlag, Berlin, 1991. 2 F. Durst and I. Benveniste, Handb. Exp. Pharmacol. 105, 293 (1993). 3 p. Bolwell, K. Bozak, and A. Zimmerlin, Phytochemistry 37, 1491 (1994). 4 D. W. Russell, J. Biol. Chem. 246, 3870 (1971). 5 I. Benveniste and F. Durst, C. R. Hebd. Seances Acad. Sci., Ser. D 278, 1487 (1974). 6 M. Potts, R. Weklych, and E. E. Conn, J. Biol. Chem. 249, 5019 (1974). 7 B. Gabriac, D. Werck-Reichhart, H. G. Teutsch, and F. Durst, Arch. Biochem. Biophys. 288, 302 (1991). 8 H. G. Teutsch, M.-P. Hasenfratz, A. Lesot, C. Stoltz, J.-M. Gamier, J.-M. Jeltsch, F. Durst, and D. Werck-Reichhart, Proc. Natl. Acad. Sci. U.S.A. 90, 4102 (1993). 9 p. Urban, D. Werck-Reichhart, H. G. Teutsch, F. Durst, S. Regnier, M. Kazmaier, and D. Pompon, Eur. J. Biochem. 222, 843 (1994).
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lignin and suberin, pigments, aroma, defense molecules (phytoalexins), antioxidants, and U V protectants. Many of these compounds are specific to certain plant families or species, and a number of other P450 enzymes are involved in these reactions. 1-3 The major difference between C A 4 H and most other plant P450s which appear to be involved in species specific reactions is that C A 4 H is probably present in all plants and in almost all tissues. Assaying CA4H is therefore a foolproof way to ascertain that microsomal preparations contain intact and active P450 electron transfer chains, and provides a sort of internal standard when extraction conditions are modified. This chapter covers the following topics: (i) the nature of the enzyme source (plant species and type of tissue), (ii) induction of CA4H activity, (iii) preparation of microsomes, and (iv) two reliable and simple assays. N a t u r e of Plant a n d T i s s u e Preparing microsomes from adult green tissues should be avoided when possible. At this stage, cells contain an enormous vacuole, and high mechanical forces are required to break the rigid walls. This leads to intensive mixing of membrane fractions with pigments and phenolics: activities are weak and difference spectra are marred by a high absorbance background and spurious redox shifts. It is better to use young (2-8 days) etiolated seedlings, root or tuber slices, and cell suspension cultures. Cell cultures are beyond the scope of this chapter. We will briefly describe a standard procedure to grow Vicia seedlings and prepare Jerusalem artichoke tuber slices. Seedlings Vicia sativa seeds are immersed for 10 min in a 0.3% sodium hypochlorite solution, washed with running tap water and distilled water, and soaked for 6 hr in distilled water at 27 °. Seeds are then sown on six layers of watersoaked filter paper placed in covered 20 x 20-cm plastic boxes and incubated for 4 days at 26 ° in total darkness. Seedlings may be used as such or further induced. In any case, the seed teguments that are rich in tannins and other polyphenols are removed manually prior to microsome preparation. Tuber Slices Reproduction of Helianthus tuberosus is vegetative. We use a clone from variety 'blanc commun' isolated in 1948, which ensures great genetic homogeneity. Other varieties purchased from stores or markets, however, show similar activities. Tubers are harvested in the fall and stored in open
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polyethylene bags in darkness at 4 °. In the case of storage tissues like tubers, C A 4 H is very low in intact tissue and is strongly enhanced by slicing, i.e., wounding, and by chemical treatments (see below). After harvest, a 3-week vernalization period is required before consistent and reproducible results are obtained.
I n d u c t i o n of CA4H C A 4 H is a highly inducible enzyme, 1,3 and since the reaction product, p-coumarate, is a precursor to so many different compounds and involved in different processes, the enzyme is induced by a wide range of factors. This may be of great help in characterizing the activity in rare or difficult to sample tissues. On the other hand, it implies that strict standardization of growth and handling conditions is necessary to achieve reproducibility.
Wounding, Infection, and Light Any kind of mechanical wounding or exposure to fungi, microbes, or viruses elicits the production of phytoalexins and tissue repair mechanisms. C A 4 H is involved in both. The elicitation of C A 4 H with fungal extracts will not be treated here, but it should be noted that an unnoticed infection may cause important activity variations. Wound Induction of H. tuberosus Tuber. Tubers are peeled, washed with distilled water, and cut in 1-mm-thick slices. Slices are washed for 2 hr in running water, rinsed with distilled water, and aged in a 2-liter Erlenmeyer flask containing 1.5 liter distilled water for 50-120 g tissue. The slices are aerated by a 4-liter min -1 stream of hydrated and filtered air, and aged in the dark at 25-30 °. It is important to consider that induction is a rapid and transient phenomenon, which reaches a peak after 18-24 hr and declines thereafter. 1° Long storage of the tubers at 4 ° before use (i.e., release of dormancy) delays and lowers maximum activity. Timing of tissue sampling for microsome preparation is thus essential for adequate activity and reproducibility. Light Is a Known Inducer of CA4H Activity. This effect is mediated by the phytochrome photoreceptor, u which may be activated either by continuous irradiation with low intensity far-red (730 nm) light or pulses of red (660 nm) light. U n d e r both conditions, C A 4 H is considerably enhanced, while chlorophyll synthesis remains minimal. As with other induction systems, it is essential to establish a time course of induction first, so sampling a0I. Benveniste, J.-P. Salaiin, and F. Durst, Phytochemistry16, 69 (1977). aa I. Benveniste, J.-P. Sala0n, and F. Durst, Phytochemistry17, 359 (1978).
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of the seedlings is made at the plateau for activity measurements. Red light induces C A 4 H in the seedlings of many plant species, but does not increase the enzyme activity in tuber slices. Seedlings are first grown for 4-7 days in the dark before irradiation. For irradiation, a projector equipped with a 660-nm interference filter is automatically switched on for 2 min every 6 hr. Even a single 5-min red pulse followed by 18 hr darkness before preparation of microsomes may double C A 4 H activity, n Chemicals
Early work from our laboratory had demonstrated that C A 4 H activity was increased when tuber slices or seedlings were exposed to certain chemicals like 2,4-dichlorophenoxyacetic acid, 12 several herbicides, phenobarbital, ethanol or to manganese ions, a3 and by aminopyrine. TM The response of C A 4 H to these compounds varies considerably depending on plant species and tissue. For example, MnC12, which increases strongly the activity in tuber slices, 13 had deleterious effects on pea seedlings. We will describe the induction of C A 4 H in H. tuberosus slices. The H. tuberosus tuber is sliced, washed, and aged in aerated water in the dark as described earlier for wound induction. Chemicals are dissolved directly into the aging water or, if needed, in a small volume of dimethyl sulfoxide added to the aging medium. The most effective inducers are 25 m M MnCI2, 20 m M aminopyrine, and 4 m M phenobarbital. The p H of the MnCI2 solution is adjusted to 7 with NaOH. As noted previously, time course studies are required to determine the peak of C A 4 H activity. For the previously mentioned inducers, maxima are observed at 48 to 72 hr after slicing. Chemical induction of 3- to 5-day-old seedlings can be achieved in a similar way. After removal of the seed tegument, seedlings are aged in an aqueous solution as described for H. tuberosus slices.
P r e p a r a t i o n of Microsomes Standard Extraction Buffer for Tuber Slices
Just before extraction, add 100 m M sodium phosphate buffer, p H 7.4, containing I m M E D T A and 250 m M sucrose; 15 m M 2-mercaptoethanol, 40 m M sodium ascorbate. Check p H after adding sodium ascorbate. 12p. Adel6, D. Reichhart, J.-P. Salatin,I. Benveniste,and F. Durst, PlantSci. Lett. 22, 39 (1981). 13D. Reichhart, J.-P. Salaiin, I. Benveniste, and F. Durst, Plant Physiol. 66, 600 (1980). 14R. Fonn6-Pfister, A. Simon, J. P. Salaiin, and F. Durst, Plant Sci. 55, 9 (1988).
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Standard Extraction Buffer for V. sativa Seedlings Just before extraction, add 100 m M sodium phosphate buffer, p H 7.4, containing 1 m M E D T A and 10% glycerol (v/v); 15 m M 2-mercaptoethanol, 40 m M sodium ascorbate, 10% (w/w of fresh tissue) insoluble PVP, and 1 m M phenylmethylsulfonyl fluoride (from a stock solution in methanol).
Additions Modifications of the extraction buffer may improve enzyme recovery, depending on plant materials. If the extraction medium turns brown after grinding with tissues and 2-mercaptoethanol no longer "smells," increase concentration or add dithiothreitol or glutathione. Significant improvement can be achieved by adding: 1% (w/v) bovine serum albumin 0.6% (w/v) Amberlite XAD-4, 10 m M NaS2Os, and 1% (w/v) insoluble PVP as described for the preparation of enzymes involved in terpene synthesis as 0.5% Dowex 1 × 2 (w/fresh weight) or pulverulent active charcoal (1-3% w/fresh weight) Dowex and charcoal are very effective in removing inhibitory polyphenols but they also adsorb protein! The effects of several of these and other additives have been recently compared. 16
Grinding and Centrifugation All operations are performed on ice. Tuber slices or Vicia seedlings are homogenized in 1.5 volume chilled extraction buffer with a Warring blender or with an Ultra-Turrax at 8000 rpm. Homogenates are filtered through a 50-/zm nylon mesh and centrifuged for 15 min at 10,000 g. The supernatant is then centrifuged for 60 min at 100,000 g. Resuspending the microsome pellet in 100 m M sodium pyrophosphate buffer containing 10 m M 2-mercaptoethanol and running a second centrifugation may improve specific activity, but it decreases C A 4 H recovery. If an ultracentrifuge is not available or if large volumes are to be handled, add MgC12 (from a 1 M stock solution) to the 10,000-g supernatant to a final concentration of 30 mM, stir for 20 min, and centrifuge for 20 min at 40,000 g. The yield is lower, but specific activities are sometimes higher using this method. 15I. M. Rajaonariviny,J. Gershenzon,and R. Croteau, Arch. Biochem. Biophys. 296,49 (1992). 16A. E. Haack and N. E. Balke, Pestic. Biochem. Physiol. 50, 92 (1994).
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The 100,000-g pellet (or the MgC12 precipitation pellet) is resuspended in sodium phosphate, p H 7.4, containing 1.5 m M 2-mercaptoethanol and 30% (v/v) glycerol to a final concentration of 5-7 mg microsomal protein m1-1. If stored at - 2 0 °, activity is stable for at least 2 months. Two CA4H Assays The two methods routinely used in our laboratory are described. H P L C determination has also been used successfully, but is more time-consuming. 9,17
Using Radiolabeled Cinnamic Acid The use of [3-14C]trans-cinnamic acid (Isotopchim, B.P. 16, F-04310 Ganobie, France) provides an easy and sensitive assay for CA4H. A 4-/.~Ci m1-1 [3-14C]cinnamate stock solution is prepared in 100 m M sodium phosphate, p H 7.4, and is stored as 2-ml aliquots at - 2 0 °. The exact cinnamate concentration of this stock solution is determined from its absorbance at 268 nm using the eM: 20,400. Depending on the C A 4 H activity in the microsomes to be studied, different amounts of cold substrate are added to this radiolabeled stock. A final cinnamate concentration ranging from 75 to 200/~M is often needed to ensure reaction linearity over 10-20 min with microsomes from induced plant tissues. Time (2 to 30 min) and protein concentration (0.05 to 0.5 mg in 200 /zl) are adjusted such that less than 50% of substrate is converted, ensuring saturation of the enzyme: the Km for cinnamate is in the 1 - 8 / ~ M range, depending on enzyme source. A control reaction, with HC1 added before N A D P H , is run to obtain the background and to make sure no contaminant running with an R~ comparable to that of coumarate is present in the substrate (it may happen occasionally). A typical assay with induced H. tuberosus microsomes contains 175 ~ M trans-cinnamic acid 0.5 m M N A D P H 0.1 mg microsomal protein 200 ~1 final sodium phosphate buffer 0.1 M, pH 7.4 When assays are incubated longer than 10 min, an N A D P H regeneration system (10 m M glucose 6-phosphate and 0.5 units glucose-6-phosphate dehydrogenase) is added to the incubation mixture. As the addition of N A D P H to plant microsomes usually results in rapid P450 inactivation, the reaction is started by the addition of N A D P H . After a 10-min incubation at 27 °, the reaction is stopped with 20 ~1 4 N HCI, and 17C. B. Stewart and M. A. Schuler, Plant Physiol. 90, 534 (1989).
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10 /xl of a mixture of nonlabeled cinnamic and coumaric acids (100/xg each) is added. A 100-/xl aliquot of reaction medium is spotted onto a precoated TLC silica gel plate 60 F254 (Merck) and is developed in the organic phase from a toluene/acetic acid/water (6/7/3) mixture. Rf is 0.54 for cinnamate and 0.3 for para-coumarate. Activity is directly determined with a thin layer analyzer (Berthold LB 2820-1). Alternatively, para-coumarate and cinnamate are visualized as purplish-blue fluorescent spots under 254-nm UV light, and silica is scraped and counted in a 5-ml Ready organic scintillation cocktail (Beckman). The reaction is usually linear for at least 10 min, but the enzyme is inactivated at incubation times over 20 rain at 27° or 10 min at 30°. The optimal pH for CA4H is around 7.4, with half-maximal activities at pH 6.2 or 8. A significant activity increase can be achieved by increasing the ionic strength of the incubation buffer (i.e., using 100 mM rather than 25 mM sodium phosphate or 100 mM Tris buffer). For the yeast-expressed enzyme, inhibition was observed when the CA4H concentration was increased above 6 pmol in the assay, is
Fluorescent Determination of 2-Naphthoate Hydroxylation Radioactivity measurements are not always possible or desired. An alternative, stemming from our recent studies of the substrate specificity of yeast expressed CA4H, TM is a very fast and sensitive fluorimetric method, a9 We have demonstrated that 2-naphthoic acid perfectly mimics transcinnamate in the CA4H active site and is metabolized with an efficiency (Km and Vmax)comparable to that of cinnamate. The product of the reaction, 6-hydroxy-2-naphthoate, is strongly fluorescent. This property can be used for a rapid but reliable assay of the CA4H activity. The optimal excitation wavelength of 6-hydroxy-2-naphthoate varies slightly with the composition of the buffer and with the concentration of NADPH and substrate in the assay. In 100 mM sodium phosphate, pH 7.4, and in the presence of 50/xM NADPH, its maximum excitation is 299 nm, with an emission peak at 443 nm. The substrate does not interfere with the readings, and a direct and continuous measurement of fluorescence in the incubation medium is possible. However, this assay cannot be used with the microsomes contaminated with pigments (e.g., chlorophyll). A typical fluorescent CA4H assay contains 100/~M 2-naphthoic acid (Sigma) 18 M. Pierrel, Y. Batard, M. Kazmaier, C. Mignotte-Vieux, F. Durst, and D. Werck-Reichhart, Eur. J. Biochem. 224, 835 (1994). 19 M. Schalk, Y. Batard, A. Seyer, A. Zimmerlin, F. Durst, and D. Werck-Reichhart, to be published.
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50 t i M N A D P H 1 m M glucose 6-phosphate 0.5 units glucose-6-phosphate dehydrogenase 0.1-0.5 mg microsomal protein 2 ml final sodium phosphate 100 mM, p H 7.4 The fluorescence of the reaction mixture without N A D P H is recorded for approximately 3 min to allow temperature equilibration (30 °) and to check for the activity in the absence of N A D P H (this step can be shortened by using a preheated buffer). N A D P H is then added to the reaction medium, and the increase in fluorescence (excitation: 299 nm; emission: 443 nm) is recorded for an additional 2 to 5 min. The reaction is usually linear for at least 10 min. Calibration of the assay is p e r f o r m e d using known amounts of 6-hydroxy-2-naphthoate (Lancaster Synthesis, Morecambe, UK). Some Typical CA4H Activities Enzyme source H. tuberosus
tuber
Wheat seedlings Vicia seedlings
Treatment Dormant Slicing + 24 hr aging Slicing + 56 hr aging in 25 mM MnC12 5 days old 4 days old 4 days old + 48 hr aging in aminopyrine
Activity (pmol sec i rag-l) 0-1 40 200 2 1 6
W h e n All A t t e m p t s H a v e Failed As already discussed, with some plants it might be impossible or very difficult to obtain measurable and reproducible C A 4 H activity. It is our experience that C A 4 H protein or transcript levels measured by Western or Northern blotting do provide a reasonable estimate of C A 4 H activity even when working with green tissues from nonlaboratory plants, i.e., plants collected in the natural environment. 2° However, the use of antibodies requires the preliminary purification of a small amount of h o m o g e n e o u s enzyme, not an easy task, although several purification methods have been published. 7,21,22 It is further hampered, at least for use on Western blots, by their p o o r cross-reactivity when the boundaries of plant families are 20y. Batard, Ph.D. Thesis, Universit6 Louis Pasteur, Strasbourg, (1995). 21M. Mizutani, D. Ohta, and R. Sato, Plant Cell PhysioL 34, 481 (1993). 22D. Werck-Reichhart, I. Benveniste, H. Teutsch, F. Durst, and B. Gabriac, AnaL Biochem. 197, 125 (1991).
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crossed. 23 This makes cDNA probes particularly attractive. CA4H has now been cloned from a range of species: H. tuberosus (Z17369), Phaseolus aureus (L07634), Medicago sativa (Ll1046), Catharanthus roseus (X69788, Z32563), A rab idopsis thaliana (T04086), Pisum sativum (U29243), and Zinnia elegans (U19922). These may be obtained from the authors or recloned using PCR or RT-PCR methods. CA4H is well conserved (generally over 80% positional nucleotide identity between different species) and crosshybridization on Northern blot seems more extended than with Western blotting. 23 D. Werck-Reichhart, Y. Batard, G. Kochs, A. Lesot, and F. Durst, Plant Physiol. 102, 1291 (1993).
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Isolation of Plant and Recombinant B y B A R B A R A A N N HALK1ER, O L E SIBBESEN,
CYP79 and
B I R G E R LINDBERG M t 0 L L E R
Introduction We have developed an isolation procedure for CYP79, also called P450tyr, which is a key enzyme in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. x Cyanogenic glucosides are secondary plant products which, on hydrolysis, release HCN. CYP79 is a multifunctional N-hydroxylase converting tyrosine into p-hydroxyphenylacetaldoxime by two consecutive N-hydroxylations, of which the first produces N-hydroxytyrosine and the second N,N-dihydroxytyrosine. 2 The latter dehydrates to 2-nitroso-3-(p-hydroxyphenyl)propionic acid which decarboxylates into the oxime. The multifunctional property of CYP79 has been confirmed by reconstitution experiments using recombinant CYP79 expressed at high levels in Escherichia coli. 3 This chapter focuses on the generally applicable procedures developed for the isolation of plant and recombinant CYP79. In addition, a strategy is suggested for successful expression of microsomal P450s in E. coli. 1 0 . Sibbesen, B. Koch, B. A. Halkier, and B. L. MOiler, Proc. Natl. Acad. Sci. U.S.A. 91, 9740 (1994). 2 0 . Sibbesen, B. Koch, B. A. Halkier, and B. L. M¢ller, J. Biol. Chem. 270, 3506 (1995). 3 B. A. Halkier, H. L. Nielsen, B. Koch, and B. L. Mealier, Arch. Biochem. Biophys. 322, 369 (1995).
METHODS IN ENZYMOLOGY,VOL. 272
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