(Received for publication, October 18, 1994)
From the
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-
-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.
Seedlings of Sorghum bicolor (L.) Moench contain large
amounts of the cyanogenic glucoside dhurrin
(-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 (
)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.
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.
[
C]L-tyrosine and
[
C](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
value of 0.14 mM and a V
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-
C]L-tyrosine (0.5 µCi), 50
µg of lipid, and 2.5 µmol KP
, pH 7.9.
[U-
C]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-
C]L-tyrosine (0.5 µCi in each
experiment), 165 pmol of NADPH, ±750 nmol of NaCl, and 2.5
µmol of KP
, 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
- 8.1
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 (
A
) 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
, 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
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.
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
[O]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
[
-
H]tyrosine as substrate for the microsomal
system, the
-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 O
-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 value is obtained in
the absence of NaCl. On the other hand, the K
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
value of 0.14 mM obtained after optimization of the
reconstitution procedure should be compared with a K
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
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.