©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome P-450 Is a Multifunctional Heme-Thiolate Enzyme Catalyzing the Conversion of

L

-Tyrosine to p-Hydroxyphenylacetaldehyde Oxime in the Biosynthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor (L.) Moench (*)

(Received for publication, October 18, 1994)

Ole Sibbesen Birgit Koch Barbara Ann Halkier Birger Lindberg Møller (§)

From the Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cytochrome P-450, which catalyzes the N-hydroxylation of L-tyrosine in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench has recently been isolated (Sibbesen, O., Koch, B., Halkier, B. A., and Møller, B. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 92, 9740-9744 ). Reconstitution of the enzyme activity in lipid micelles containing cytochrome P-450 and NADPH-cytochrome P-450 oxidoreductase demonstrates that cytochrome P-450 catalyzes the conversion of L-tyrosine into p-hydroxyphenylacetaldehyde oxime. Earlier studies with microsomes have demonstrated that this conversion involves two N-hydroxylation reactions of which the first produces N-hydroxytyrosine. We propose that the product of the second N-hydroxylation reaction is N,N-dihydroxytyrosine. N,N-dihydroxytyrosine is dehydrated to 2-nitroso-3-(p-hydroxyphenyl) propionic acid which decarboxylates to p-hydroxyphenylacetaldehyde oxime. The dehydration and decarboxylation reactions may proceed non-enzymatically. The E/Z ratio of the p-hydroxyphenylacetaldehyde oxime produced by reconstituted cytochrome P-450 is 69:31. Lipid micelles made from L-alpha-dilauroyl phosphatidylcholine are more than twice as effective in reconstituting cytochrome P-450 activity as compared to other lipids. The K and turnover number of the enzyme is 0.14 mM and 200 min, respectively, when assayed in the presence of 15 mM NaCl whereas the values are 0.21 mM and 230 min when assayed in the absence of added salt. The multifunctional nature cytochrome P-450 is confirmed by demonstrating that binding of L-tyrosine or N-hydroxytyrosine mutually excludes binding of the other substrate. These results explain why the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime as earlier reported (Møller, B. L., and Conn, E. E.(1980) J. Biol. Chem. 255, 3049-3056) shows the phenomenon of catalytic facilitation (``channeling''). Cytochrome P-450 is the first isolated multifunctional heme-thiolate enzyme from plants. N-Hydroxylases of the cytochrome P-450 type with high substrate specificity have not previously been reported.


INTRODUCTION

Seedlings of Sorghum bicolor (L.) Moench contain large amounts of the cyanogenic glucoside dhurrin (beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) (1) . The key intermediates in the biosynthesis of dhurrin are N-hydroxytyrosine, p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile(2, 3, 4, 5) . The pathway has been elucidated through biosynthetic studies using microsomes isolated from etiolated sorghum seedlings. The microsomal fraction catalyzes the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile(2, 4) , which in vivo is glucosylated into dhurrin by a soluble UDPG-glucosyltransferase(6) . Three hydroxylation steps consuming three molecules of oxygen have been identified in the conversion of tyrosine to p-hydroxymandelonitrile(7) . Two of these oxygen molecules are consumed in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime(7) . The involvement of a monooxygenase of the cytochrome P-450 (heme thiolate) type in the conversion of tyrosine to N-hydroxytyrosine has previously been reported(8) . Cytochrome P-450 monooxygenases consist of a NADPH-cytochrome P-450 oxidoreductase transferring reducing equivalents from NADPH to cytochrome P-450, which is the substrate binding and catalytic component of the monooxygenase(9) . A heme-thiolate enzyme designated cytochrome P-450 has recently been isolated from microsomes prepared from etiolated seedlings of S. bicolor (L.) Moench(10, 11) . Cytochrome P-450 forms a Type I substrate binding spectrum with L-tyrosine (10, 11) , and a polyclonal antibody raised against the isolated enzyme inhibits the ability of the microsomal system to metabolize tyrosine by 60%(10) . From these results, isolated cytochrome P-450 was inferred to catalyze the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of dhurrin(4) . Cytochrome P-450 is the first enzyme specific to the biosynthesis of cyanogenic glucosides to be isolated and characterized.

In the present paper, we have reconstituted the cytochrome P-450 monooxygenase into micelles of DLPC (^1)and demonstrate that cytochrome P-450 catalyzes the conversion of L-tyrosine all the way to p-hydroxyphenylacetaldehyde oxime. The multifunctional nature of cytochrome P-450 is confirmed by showing that L-tyrosine and N-hydroxytyrosine are mutually exclusive substrates for cytochrome P-450 as monitored by substrate binding spectra and sequential administration of the two substrates.


MATERIALS AND METHODS

Chemicals

DEAE-Sepharose fast flow, 2`,5`-ADP Sepharose 4B, Sephadex G-50, and Sephacryl S-100 were purchased from Pharmacia Biosystems, Uppsala, Sweden. Cibacron Blue Agarose and Reactive Red 120 Agarose were obtained from Sigma. Reduced Triton X-100 was from Aldrich, Steinheim, Germany, and RENEX 690 from J. Lorentzen A/S, Kvistgård, Denmark. N-Hydroxytyrosine was synthesized from L-tyrosine(12) . All other chemicals used were of reagent grade and purchased from Sigma. [U-^14C]L-Tyrosine (450 mCi/mmol) was obtained from Amersham Ltd. [U-^14C]-p-Hydroxyphenylacetaldehyde oxime was synthesized from [U-^14C]L-tyrosine using the sorghum microsomal system prepared in the absence of dithiothreitol(4, 13) .

Enzyme Assays

Quantitative determination of total cytochrome P-450 was carried out by difference spectroscopy using an extinction coefficient of 91 cm mM for the adduct between reduced cytochrome P-450 and carbon monoxide(14) . Cytochrome P-450 substrate binding spectra were recorded on SLM-Aminco DW-2C and DW-2000 spectrophotometers according to Jefcoate(15) .

Isolation of Cytochrome P-450

Isolation of the cytochrome P-450 enzyme was performed essentially as described previously (10) with the following modifications. After elution of the cytochrome P-450 from the Reactive Red Agarose 120 column, the eluate was gel filtrated through a Sephadex G-50 column, equilibrated in a buffer composed of 50 mM KP(i), pH 7.9, 400 mM KCl, 0.1% CHAPS, 2 mM DTT. Eluted cytochrome P-450 was dialyzed for 2 h against 50 mM KP(i), pH 7.9, 2 mM DTT, diluted 4-fold with dialysis buffer in an Amicon ultrafiltration cell fitted with an YM-30 membrane, and concentrated to 1.45 nmol/ml.

Purification of NADPH-Cytochrome P-450 Oxidoreductase

The purification of the NADPH-cytochrome P-450 oxidoreductase was modified from Halkier and Møller (8) as follows. The oxidoreductase containing eluate from the 2`,5`-ADP Sepharose 4B column was extensively dialyzed toward a buffer composed of 10 mM KP(i), pH 7.9, 1 mM DTT (dialysis buffer) until the concentration of KCl was less than 10 mM as monitored by conductivity measurements. The dialyzed sample was applied to a DEAE-Sepharose Fast Flow column (5 times 50 mm), equilibrated in dialysis buffer fortified with 0.1% RENEX 690. The column was washed with 20 ml of dialysis buffer. The RENEX 690 was exchanged with CHAPS by washing the column with 20 ml of dialysis buffer fortified with 0.1% CHAPS. The NADPH-cytochrome P-450 oxidoreductase was eluted with a 0-1 M linear KCl gradient (2 times 5 ml) prepared in dialysis buffer fortified with 0.1% CHAPS. A major part of the oxidoreductase was reversibly inactivated during the purification procedure and was reactivated by addition of DTT to a final concentration of 8 mM and subsequent incubation for 30 min at room temperature. After incubation, the sample was diluted 4-fold with 50 mM KP(i), pH 7.9, and concentrated in an Amicon ultrafiltration cell fitted with an YM-30 membrane to a final activity of 15 units/ml where 1 unit is defined as the amount of NADPH-cytochrome P-450 oxidoreductase which reduces 1 µmol of cytochrome c/min at 25 °C in an assay mixture containing 50 mM KP(i), pH 7.9, 0.05 mM cytochrome c, and 0.25 mM NADPH(16) .

Reconstitution of Cytochrome P-450

Lipid (10 mg/ml) was suspended in 50 mM KP(i), pH 7.9, by sonication (1 min) using a Branson Sonifier 250 equipped with a 3-mm microtip, set at lowest intensity. The lipid suspension (10 µl) was mixed in a glass vial with NADPH-cytochrome P-450 oxidoreductase (0-0.15 unit), cytochrome P-450 (0-1.5 pmol) and adjusted to the desired final volume with 50 mM KP(i), pH 7.9. [U-^14C]L-tyrosine (10 µl, 0.5 µCi) or [U-^14C]L-p-hydroxyphenylacetaldehyde oxime (10 µl, 0.5 µCi) were tested as substrates. NADPH (10 µl, 25 mg/ml) was added as electron donor. The reaction mixture was then sonicated for 1 min using a Branson 5200 sonication bath and incubated at 30 °C. The reaction was stopped by transferring the glass vials onto ice. The radioactively labeled intermediates formed were extracted into 50 µl of ethyl acetate and quantified by TLC/autoradiography (8) or by HPLC analysis/solid state scintillation counting (13) as previously reported, except that the isocratic elution of the reverse phase column was performed using a mobile phase composed of 10% 2-propanol in 1 mM Tricine (pH 7.9).


RESULTS

The heme-thiolate enzyme cytochrome P-450 was isolated as described previously(10, 11) . For use in reconstitution experiments, a gel filtration step was included to lower the CHAPS concentration followed by a dilution/concentration step to reduce the concentration of KCl from 400 mM to less than 10 mM. In the preparation of the NADPH-cytochrome P-450 oxidoreductase, a small ion-exchange column was introduced to exchange detergent from RENEX 690 to CHAPS and as a concentration step. The activity of the oxidoreductase was increased severalfold by a DTT treatment. In reconstitution experiments, the two membrane proteins were added to micelles of DLPC and subjected to gentle sonication to facilitate membrane fusion. [^14C]L-tyrosine and [^14C](E,Z)-p-hydroxyphenylacetaldehyde oxime were used as substrates. Two different analytical procedures were used. The HPLC method permits the separation of (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime (Fig. 1) whereas in the TLC system the (E)- and (Z)-oxime isomerize rapidly and comigrate (Fig. 2). The TLC system is advantageous in being more sensitive compared to the HPLC procedure. When reconstituted into micelles of DLPC, cytochrome P-450 catalyzes the conversion of L-tyrosine all the way to p-hydroxyphenylacetaldehyde oxime ( Fig. 1and Fig. 2). No conversion is seen in those reaction mixtures from which NADPH (Fig. 1) or NADPH-cytochrome P-450 oxidoreductase (Fig. 2) is excluded. The formation of p-hydroxyphenylacetaldehyde oxime (Fig. 1) demonstrates that cytochrome P-450 is a multifunctional heme-thiolate protein catalyzing reactions in addition to the initial N-hydroxylation of tyrosine. Using the TLC-autoradiography system, minute amounts of radiolabeled products comigrating with authentic p-hydroxybenzaldehyde and 1-nitro-2-(p-hydroxyphenyl) ethane are detected in the reaction mixtures.


Figure 1: The catalytic properties of reconstituted cytochrome P-450 as analyzed by reverse phase HPLC in the presence or absence of NADPH. The elution profiles obtained by continuously monitoring the absorbance at 280 nm (A) and the radioactivity content of the effluent (C-14) are shown. Inset A, superimposed 280 nm elution profile of authentic compounds. 1, p-hydroxyphenylacetonitrile; 2, (E)-p-hydroxyphenylacetaldehyde oxime; 3, (Z)-p-hydroxyphenylacetaldehyde oxime; 4, 1-nitro-2-(p-hydroxyphenyl)ethane.




Figure 2: The catalytic properties of reconstituted cytochrome P-450 as analyzed by TLC chromatography using radiolabeled L-tyrosine or p-hydroxyphenylacetaldehyde oxime as substrates in the presence or absence of NADPH-cytochrome P-450 oxidoreductase. The chromatographic mobility of authentic standards is indicated.



Different lipids were tested for their ability to reconstitute the cytochrome P-450 monooxygenase (Fig. 3). DLPC as used in the experiments described above was found to be more than twice as effective as any of the other lipids tested. Metabolism of L-tyrosine to p-hydroxyphenylacetaldehyde oxime was observed even in the absence of added lipid. Using DLPC for the reconstitution experiments, the optimum concentration of NaCl was found to be 15 mM (Fig. 4). In the presence of 15 mM NaCl in the reconstitution assay, a K(m) value of 0.14 mM and a V(max) value of 198 turnovers min was obtained. The corresponding values in the absence of added NaCl were 0.21 mM and 228 turnovers min.


Figure 3: The effect of different lipids on reconstitution of cytochrome P-450. The assay mixtures (50 µl) contained 1.45 pmol cytochrome P-450, 0.15 unit of NADPH-cytochrome P-450 oxidoreductase, 50 nmol of [U-^14C]L-tyrosine (0.5 µCi), 50 µg of lipid, and 2.5 µmol KP(i), pH 7.9. [U-^14C]L-Hydroxyphenylacetaldehyde oxime formed was quantified using HPLC and solid state glass scintillation counting.




Figure 4: Lineweaver-Burk plot showing the effect of NaCl on the kinetics of the cytochrome P-450 enzyme. The assay mixture (50 µl) contained 1.3 pmol of cytochrome P-450, 0.15 unit of NADPH-cytochrome P-450 oxidoreductase, 50 µg of DLPC, 0-12 nmol of [U-^14C]L-tyrosine (0.5 µCi in each experiment), 165 pmol of NADPH, ±750 nmol of NaCl, and 2.5 µmol of KP(i), pH 7.9.



p-Hydroxyphenylacetaldehyde oxime exists in two geometric forms, the E- and Z-isomers. The two isomers are relatively stable in the buffer system used with less than 5% isomerization taken place after 30 min of incubation(13) . An E/Z equilibrium ratio of 58:42 is reached upon prolonged standing(13) . To determine the ratio between the oxime isomers produced by cytochrome P-450, the reconstituted monooxygenase was incubated with tyrosine as substrate for different time periods at the end of which the two isomers were quantified by HPLC analysis (Fig. 5). Both isomers were present at all time periods tested with a slight decrease in the E/Z ratio upon prolonged incubation. The percentage of Z-isomer of the total amount of oxime may be fitted to the exponential function % Z-oxime = 39.5 - 8.1bullet e, where x is the incubation time in minutes. Extrapolation of the data predicts that the E/Z ratio initially produced by the monooxygenase is 69:31 whereas the thermodynamic equilibrium ratio achieved under these experimental conditions is 60:40.


Figure 5: The isomer of p-hydroxyphenylacetaldehyde oxime formed by the reconstituted cytochrome P-450 enzyme as a function of the incubation time. Assay conditions are as in legend to Fig. 3.



Cytochrome P-450 has previously been reported to produce a Type I substrate binding spectrum in the presence of L-tyrosine(10, 11) . In accordance with the ability of the isolated enzyme to catalyze the formation of p-hydroxyphenylacetaldehyde oxime upon reconstitution, its ability to bind N-hydroxytyrosine was tested (Fig. 6). A substrate binding spectrum identical to that obtained with L-tyrosine was obtained using N-hydroxytyrosine as substrate. When a saturating amount of N-hydroxytyrosine was added to isolated cytochrome P-450 already saturated with L-tyrosine or vice versa, the size of the substrate binding spectrum (DeltaA) remained unchanged. This demonstrates that the two substrates are competing for the same active site and confirms that cytochrome P-450 is a multifunctional enzyme. From the amounts of cytochrome P-450 used, the absorption coefficient () is calculated to 67 cm mM. A complete transition from a low spin state to a high spin state would have resulted in an absorption coefficient of 138 cm mM(15) .


Figure 6: Substrate binding spectra of cytochrome P-450. Each cuvette contained (final volume, 500 µl) 25 µmol of KP(i), pH 7.9, cytochrome P-450 (0.32 nmol in experiments A and B, 0.57 nmol in experiments C and D). Substrates (5 mM tyrosine or 20 mMN-hydroxytyrosine) were added to the measuring cuvette as indicated. Identical volumes of 50 mM KP(i) were added to the reference cuvette. A, titration with tyrosine (tyr); B, saturation with tyrosine, then saturation with N-hydroxytyrosine; C, titration with N-hydroxytyrosine; D, saturation with N-hydroxytyrosine, then saturation with tyrosine.




DISCUSSION

A priori, reconstituted cytochrome P-450 was expected to convert L-tyrosine into N-hydroxytyrosine, which has previously been shown to be the first intermediate in dhurrin biosynthesis(4) . Surprisingly, the reconstitution experiments demonstrate that cytochrome P-450 is multifunctional catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime, i.e. the same conversion as catalyzed by sorghum microsomes prepared in the absence of a reducing agent(2, 17) . As discussed below, the demonstrated multifunctionality of cytochrome P-450 gives rize to new interpretations of the results obtained in previous biosynthetic experiments concerning the identity and number of intermediates involved in the biosynthetic pathway and provides a biochemical mechanism by which the previously reported ``channeling phenomenon'' (17) can be explained.

Initial elucidation of the biosynthetic pathway for cyanogenic glucosides involved in vivo administration of radioactively labeled amino acids to excised plant parts(18, 19) . Efficient incorporation of radioactivity into cyanogenic glucosides was observed, but no intermediates were detectable. In vitro biosynthetic studies using microsomes demonstrated efficient conversion of amino acids to cyanohydrins with oximes as the only intermediate to accumulate in significant amounts(2, 4) . The additional compounds listed as intermediates in the pathway have been included based on in vitro trapping and feeding experiments demonstrating the ability of microsomes to catalyze the formation as well as further metabolism of the compounds (2, 3, 4, 5) (Fig. 7). Stoichiometric studies showed that two molecules of oxygen are consumed in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime(7) . Biosynthetic studies using [^18O]oxygen and tyrosine and N-hydroxytyrosine as substrates have shown that the oxygen atom of the hydroxyamino group of N-hydroxytyrosine introduced by N-hydroxylation of L-tyrosine is specifically lost in the overall conversion of N-hydroxytyrosine to p-hydroxyphenylacetaldehyde oxime whereas the second oxygen atom incorporated by N-hydroxylation of N-hydroxytyrosine is quantitatively retained in the p-hydroxyphenylacetaldehyde oxime formed(5) . The ability of the microsomes to discriminate between the two oxygen atoms introduced by the consecutive N-hydroxylation reactions precludes the possibility of free rotation around the C-N single bond as would occur if an intermediate produced by one enzyme would have to diffuse to a second enzyme before further metabolism takes place (Fig. 7).


Figure 7: The biosynthetic reactions catalyzed by the reconstituted cytochrome P-450 monooxygenase.



In light of these findings and considerations, a likely route for the formation of the oxime would be via N,N-dihydroxytyrosine (Fig. 7). This compound is extremely labile. It would dehydrate non-enzymatically to produce 2-nitroso-3-(p-hydroxyphenyl)propionate which would decarboxylate into p-hydroxyphenylacetaldehyde oxime. Cytochrome P-450 would then exert its catalytic function by catalyzing two N-hydroxylation reactions whereas the subsequent dehydration and decarboxylation reactions would not necessarily need to be enzyme catalyzed. Decarboxylation reactions have not previously been shown to involve cytochrome P-450 enzymes(20) . In previous studies using [alpha-^2H]tyrosine as substrate for the microsomal system, the alpha-hydrogen atom of tyrosine was shown to be quantitatively retained in the p-hydroxyphenylacetaldehyde oxime produced(13) . The pathway outlined in Fig. 7is in agreement with this observation.

Earlier biosynthetic experiments showed that N-hydroxytyrosine produced by the microsomal system does not freely equilibrate with exogenously added N-hydroxytyrosine thus exhibiting the phenomenon of catalytic facilitation (channeling) with facilitation ratios between 25 and 160 dependent on the experimental conditions(17) . The channeling and ^18O(2)-labeling data can now be explained by the fact that both N-hydroxylation reactions are catalyzed by a multifunctional monooxygenase with a single catalytic site in which each of the two oxygen atoms have fixed positions mediated, e.g. by hydrogen bonds, metal chelation, or by a positively charged amino acid side chain. Thus, N-hydroxytyrosine is not a genuine free intermediate and possibly best considered a stable form of a ``transition state.''

The demonstrated ability of the microsomal system to produce and metabolize 1-nitro-2-(p-hydroxyphenyl)ethane (5, 7) in combination with the consumption of two molecules of oxygen in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime (7) previously led us to the conclusion that 2-nitro-3(p-hydroxyphenyl)propionate and aci-1-nitro-2-(p-hydroxyphenyl)ethane are intermediates in the biosynthetic pathway(5, 7) . A reduction step would then be required to convert the aci-nitro compound to the oxime. 1-Nitro-2-(p-hydroxyphenyl)ethane is also produced in low amounts when L-tyrosine is administered to the reconstituted cytochrome P-450 enzyme (Fig. 2). According to the biosynthetic pathway outlined in Fig. 7, the minute amounts of nitro compound produced constitutes a side product. The nitro compound would be formed if N,N-dihydroxytyrosine or its dehydration product 2-nitroso-3-(p-hydroxyphenyl)propanoic acid is subjected to an additional N-hydroxylation reaction before leaving the catalytic site. Alternatively, a small percentage of the N,N-dihydroxytyrosine may be prone to chemical oxidation instead of dehydration. Perturbation of cytochrome P-450 during preparation of microsomes or during enzyme isolation may generate low amounts of a conformation of the enzyme which favors nitro compound formation. It is to be noted that the glucoside of 1-nitro-2-(p-hydroxyphenyl)ethane accumulates in osmotically stressed cell suspension cultures of California poppy (Eschscholtzia californica Cham.)(21) . The intact plant produces the tyrosine-derived cyanogenic glucosides triglochinin and dhurrin, which do not accumulate in the cell suspension cultures. Whether the nitro compound accumulates in intact plants under osmotic stress remains unknown. These data demonstrate that the nitro compound is easily derived from the pathway. The observed ability of the microsomal system to catalyze a low rate of cyanide formation from 1-nitro-2-(p-hydroxyphenyl)ethane may reflect an unspecific enzymatic activity associated with the microsomal membranes.

The reconstituted cytochrome P-450 enzyme produces (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime in an E/Z ratio of 69:31. In biosynthetic studies using the microsomal enzyme system, the E/Z ratio of the produced oxime varied between 70:30 and 77:23(13) . The microsomal system initially produces the (E)-isomer. The (E)-isomer is then converted to the (Z)-isomer as an obligatory step in its further conversion to p-hydroxymandelonitrile(13) . The latter information makes us propose that the (Z)-isomer is the in vivo product of cytochrome P-450 although the (E)-isomer is the major isomer formed in the two in vitro systems. Possibly, the cytochrome P-450 used in the in vitro studies has been perturbed during the isolation and reconstitution procedure resulting in an enzyme with a differently contoured active site favoring unrestricted release of the (E)-isomer from the active site before its conversion to the (Z)-isomer. Such a perturbation may also explain why relatively large amounts of the nitro compound are produced using the reconstituted system compared with the microsomal system (Fig. 2). Further studies are needed to clarify this matter. Other reconstituted cytochrome P-450 enzymes have previously been documented to possess slightly altered catalytic properties(22, 23) .

The activity of the reconstituted cytochrome P-450 is quantitatively dependent on the type of lipid used for micelle formation with DLPC providing more than twice as high rates as any of the other tested lipids. The majority of the previously reconstituted cytochrome P-450 enzymes reconstitute well with DLPC, but specific requirements for unsaturated phospholipids have also been observed (22) . In the reconstitution of cytochrome P-450, a mixture of all lipids tested is not as effective as DLPC alone indicating that other lipids may prevent proper interaction between cytochrome P-450, NADPH-cytochrome P-450 oxidoreductase, and DLPC. In contrast, the adrenal cytochrome P-450 is best reconstituted in the presence of a mixture of lipids(23) .

The kinetic properties of the reconstituted enzyme system are influenced by the NaCl concentration. Using L-tyrosine as substrate, the highest V(max) value is obtained in the absence of NaCl. On the other hand, the K(m) value is decreased from 0.21 to 0.14 mM upon addition of 15 mM NaCl indicating that a higher ionic strength facilitates substrate binding. A high ionic strength has previously been reported to facilitate electron transport from NADPH-cytochrome P-450 oxidoreductase to cytochrome P-450(16) . The K(m) value of 0.14 mM obtained after optimization of the reconstitution procedure should be compared with a K(m) value of 0.03 mM for the enzyme when assayed as a component of the microsomal system(4) . Using the microsomal enzyme system and L-tyrosine as substrate, the V(max) value is 145 nmol of hydrogen cyanide/mg protein/h(17) . In this system, the conversion of tyrosine to N-hydroxytyrosine is the rate-limiting step(5) . The total content of cytochrome P-450 enzymes in the microsomal system as determined by the carbon monoxide difference spectrum is approximately 0.2 nmol of total cytochrome P-450/mg protein of which the cytochrome P-450 enzyme constitutes about 20%(10) . The calculated turnover number of the cytochrome P-450 enzyme when present as a component of the microsomal system is therefore approximately 60 min. The turnover number of reconstituted cytochrome P-450 is 228 min. The higher turnover number obtained with the reconstituted system may reflect that the ratio between NADPH-cytochrome P-450 oxidoreductase and cytochrome P-450 is much lower in the microsomal membrane compared to the reconstituted system. At the saturating substrate conditions used, NADPH-cytochrome P-450 oxidoreductase may limit the activity of the microsomal system. The corresponding turnover numbers for cinnamic acid 4-hydroxylase are 0.055 min for the reconstituted enzyme and 20 min for the microsomal system(16) . The reported turnover numbers for reconstituted cytochrome P-450 enzymes vary considerably as further exemplified by reconstituted geraniol 10-hydroxylase isolated from Catharanthus roseus which has a turnover number of 1740 min(25) whereas reconstituted 3,9-dihydroxypterocarpan 6a-hydroxylase from Glycine max has a turnover number of 9 min(26) . Residual amounts of detergents, suboptimal choice of lipids, or inefficient interaction with NADPH-cytochrome P-450 oxidoreductase may serve to lower the measurable activity of reconstituted cytochrome P-450 monooxygenases.

In conclusion, reconstitution of the cytochrome P-450 monooxygenase has demonstrated that cytochrome P-450 is multifunctional and catalyzes the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime. The pathway for biosynthesis of cyanogenic glucosides has previously been demonstrated to be channeled(17) . The present data explain why the first half of the pathway is channeled.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Plant Biochemistry Laboratory, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark. Tel: +45-35283352; Fax: +45-35283333.

(^1)
The abbreviations used are: DLPC, L-alpha-dilauroyl phosphatidylcholine; DOPC, L-alpha-dioleyl phosphatidylcholine; DLPE, L-alpha-dilauroyl phosphatidylethanolamine; DOPE, L-alpha-dioleyl phosphatidylethanolamine; DPPS, DL-alpha-dipalmitoyl phosphatidylserine; DTT, dithiothreitol; KP(i), KH(2)PO(4)/K(2)HPO(4); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TLC, thin layer chromatography; HPLC, high performance liquid chromatography.


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