From the Plant Biochemistry Laboratory, the
Department of Chemistry, and the § Center for
Molecular Plant Physiology (PlaCe), The Royal Veterinary and
Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C,
Denmark, the ** SwissProt Group, Swiss Institute of Bioinformatics,
CH-1211 Geneva 4, Switzerland, and the
IACR-Rothamsted, Harpenden, AL5 2JQ, United
Kingdom
Received for publication, March 23, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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CYP83B1 from Arabidopsis thaliana has
been identified as the oxime-metabolizing enzyme in the biosynthetic
pathway of glucosinolates. Biosynthetically active microsomes isolated
from Sinapis alba converted
p-hydroxyphenylacetaldoxime and cysteine into
S-alkylated p-hydroxyphenylacetothiohydroximate,
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450
monooxygenase. We searched the genome of A. thaliana for
homologues of CYP71E1 (P450ox), the only known oxime-metabolizing
enzyme in the biosynthetic pathway of the evolutionarily related
cyanogenic glucosides. By a combined use of bioinformatics, published
expression data, and knock-out phenotypes, we identified the cytochrome
P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate
pathway as evidenced by characterization of the recombinant protein
expressed in Escherichia coli. The data are consistent with
the hypothesis that the oxime-metabolizing enzyme in the cyanogenic
pathway (P450ox) was mutated into a "P450mox" that
converted oximes into toxic compounds that the plant detoxified into glucosinolates.
Glucosinolates are naturally occurring amino acid-derived
S-glucosides of thiohydroximate-O-sulfonates.
They co-occur with endogenous thioglucosidases called myrosinases that
upon tissue damage hydrolyze glucosinolates into a wide range of
degradation products such as e.g. isothiocyanates, nitriles,
and thiocyanates. Glucosinolates (or rather their degradation products)
are involved in plant defense and constitute characteristic flavor
compounds and cancer-preventive agents in Brassica vegetables.
The biosynthetic pathway from precursor amino acid to the core
glucosinolate structure has been well studied, and many of the
intermediates are known, including oximes, thiohydroximic acids, and
desulfoglucosinolates (1, 2). Recently, it has been shown that
cytochromes P450 belonging to the CYP79 family catalyze the conversion
of amino acids to oximes (3-7). Little is known about the formation of
thiohydroximic acids from oximes. The remaining part of the pathway for
the core structure involves a UDP-glucose:thiohydroximic acid
glucosyltransferase and a sulfotransferase (for review, see Ref.
2).
It has been proposed that aci-nitro compounds are
intermediates in the conversion of oximes to thiohydroximic acids (8). This was supported by isolation of 1-nitro-2-phenylethane from Tropaeolum majus shoots and by in vivo conversion
of phenylacetaldoxime into 1-nitro-2-phenylethane and of
1-nitro-2-[1,2-14C]phenylethane into benzylglucosinolate
(9). The aci-nitro is proposed to be conjugated with a
sulfur donor to produce an S-alkyl thiohydroximate, possibly
by a glutathione S-transferase (2). Biochemical studies
indicate that the S-alkyl thiohydroximate is subsequently
hydrolyzed to the thiohydroximic acid by a C-S lyase (10).
Glucosinolates are related to cyanogenic glucosides because both groups
of natural plant products are derived from amino acids and have oximes
as intermediates. This suggests that the oxime-metabolizing enzyme is
the branching point between the cyanogenic glucoside and the
glucosinolate pathway. In the biosynthetic pathway of the
tyrosine-derived cyanogenic glucoside dhurrin from Sorghum bicolor, the oxime-metabolizing enzyme (designated P450ox or
CYP71E1) catalyzes the conversion of oxime to We have previously used Sinapis alba as a model plant for
biosynthetic studies of the glucosinolate pathway (13). The
tyrosine-derived p-hydroxybenzylglucosinolate is the major
glucosinolate in S. alba. In the present study, we
characterize biochemically the oxime-metabolizing enzyme in microsomes
from S. alba and show that the enzyme is dependent on a
cytochrome P450 monooxygenase. In addition, we show that CYP83B1 is the
oxime-metabolizing enzyme in glucosinolate biosynthesis in
Arabidopsis thaliana as evidenced by biochemical
characterization of the recombinant protein. The data substantiate the
results recently obtained with the rnt1-1 CYP83B1 knock-out
mutant (29).
Chemical Synthesis
S-(Benzohydroximoyl)--
L-cysteine S-(Benzohydroximoyl)-N-acetyl--
L-cysteine Preparation of Microsomal Enzyme System from S. alba
Seeds of S. alba were germinated in darkness and
jasmonate-treated as previously described (13). The preparation of
microsomes was done as previously described (13) except that the
isolation buffer was modified to consist of 250 mM Tricine,
100 mM ascorbic acid, 2 mM EDTA, and 2 mM dithiothreitol, pH 8.2. The same procedure was used to
prepare microsomes from seedlings of T. majus, S. bicolor, and Zea mays.
Measurements of Oxime-metabolizing Enzyme Activities in
Microsomes from S. alba
[U-14C]p-Hydroxyphenylacetaldoxime was
produced enzymatically by CYP79A1 as previously described (14). In a
typical enzyme assay, 1 mg of microsomal protein was incubated in
buffer containing 30 mM KPi, pH 7.9, 3 mM NADPH, 38 nCi (66 pmol) of
[U-14C]p-hydroxyphenylacetaldoxime, and 6 mM cysteine or N-acetylcysteine in a total
volume of 200 µl. After incubation at 29 °C for 1 h, the
reaction mixtures were extracted twice with 400 µl of ethyl acetate.
If N-acetylcysteine was used as a sulfur donor, the
remaining water phases were acidified by addition of 200 µl of 1%
formic acid and extracted three times with 800 µl of ethyl acetate.
The ethyl acetate phases were combined and dried in vacuo.
The extracts were redissolved in 30 µl of 50% ethanol and analyzed
by TLC. If cysteine was used as a sulfur donor 400 µl of 96% ethanol
were added to the remaining water phases, and the solution was
clarified by centrifugation for 15 min at 20,000 × g.
The supernatants were concentrated in vacuo, redissolved in
30 µl of 50% ethanol, and analyzed by TLC. Boiled microsomes were
used in control assays. For analysis of the effect of treatment with
cytochrome P450 inhibitor, standard microsomal reaction mixtures were
incubated in the presence of 0.1 mM tetcyclasis. The TLC
was performed on Silica Gel 60 F254 sheets (Merck) using
2-propanol:ethyl acetate:water (7:1:2, v/v) as eluent. The TLC plates
were eluted twice to improve separation. Radiolabeled bands were
visualized and quantified on a STORM 840 PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). For LC-MS analysis, 5 × 200 µl of
reaction mixtures were made as described above except that
[U-14C]p-hydroxyphenylacetaldoxime was
exchanged for 3 mM unlabeled oxime. The compounds were
extracted from the reaction mixtures as described above.
LC-MS Analysis
Electrospray ionization LC-MS analysis was done on a HP1100 LC
coupled to a Bruker Esquire-LC ion trap mass spectrometer. The
reversed-phase LC conditions were as follows: column: XTerra MS C18 3.5 µm, 2.1 × 100 mm (Waters Corp.); mobile phases A: 0.03% HCOOH, B: 80% MeCN, 0.1% HCOOH. The flow rate was 0.2 ml/min, and the
gradient program was 0-2 min isocratic 5% B, 2-30 min linear
gradient 5-100% B, and 30-35 min isocratic 100% B. The mass
spectrometer was run in positive ion mode using a low sampling cone
voltage to minimize fragmentation. Authentic standards of S-(benzohydroximoyl)-L-cysteine and
S-(benzohydroximoyl)-N-acetyl-L-cysteine were synthesized and used to develop conditions for the recording of
mass spectra of analogue compounds (see above).
Phylogenetic Trees
The protein sequences of all the members of the CYP71 family
were corrected for prediction errors, and the full-length sequences were aligned including one member of each of the families CYP76, CYP98,
and CYP84 using ClustalW (15). Phylogenetic analysis was performed with
the Protdist and Fitch (Fitch-Margoliash and least squares method)
programs of the Phylip package (16).
Generation of the Constructs for Escherichia coli Expression
The ESTs 226P8T7, 5G6, and 148G2T7 (Arabidopsis Biological
Resource Center, Columbus, OH), which encode the full-length
sequences of CYP71B6, CYP71B7, and
CYP83B1, respectively, were expressed heterologously in
E. coli using the pSP19 g10L expression vector (17). Silent
mutations were introduced to enrich for A and T in the first 11 codons
(17). The coding region of CYP71B6 was amplified from the
EST 226P8T7 by PCR with primer 1 (sense direction; 5'-GGAATTCCATATGTCACTTTTATCTTTCCCCATT-3') and primer 2 (antisense direction; 5'-GGCTGCAGGCATGCTTAAAGCTTGCGGTTGATGA-3'). The
PCR was set up in a total volume of 100 µl in Pwo
polymerase PCR buffer with 2 mM MgSO4 using 4 units of Pwo polymerase (Roche Molecular Biochemicals), 1.3 µg of template DNA, 200 µM dNTPs, and 100 pmol of each
primer. The PCR was incubated for 2 min at 94 °C, 21 cycles of
15 s at 94 °C, 20 s at 56 °C, and 70 s at
72 °C. The PCR fragment was digested with EcoRI and
PstI, ligated into pBluescript II SK (Stratagene), and
transferred from pBluescript II SK to an NdeI/SphI-digested pSP19 g10L vector. The
CYP71B7 gene was PCR-amplified from EST 5G6 using primer 3 (sense direction; 5'-GGAATTCATATGGCTATCTTGCTCTGTTTC-3') and primer 4 (antisense direction; 5'-CGGGATCCCATGATCGTCATCTTAATGATG-3'). The PCR
was set up as described above. The PCR was incubated for 2 min at
94 °C, 23 cycles of 15 s at 94 °C, 45 s at 55 °C,
and 2 min at 72 °C. The PCR product was digested with
EcoRI and BamHI, ligated into pBluescript II SK,
and transferred from pBluescript II SK into an
NdeI/BamHI-digested pSP19 g10L. The
CYP83B1 coding region was amplified from the EST 148G2T7 by
PCR with primer 5 (sense direction;
5'-GGAATTCCATATGAAACTCTTATTGATTATAGCTGGTTTAGTTGCGGCTGCAG-3') and
primer 6 (antisense direction; 5'-CGGGATCCATTAGATGTGTTTCGTTGGTGC-3'). The PCR was set up as described above. The PCR was incubated for 2 min at 94 °C, 22 cycles of 15 s at 94 °C, 20 s at
57 °C, and 70 s at 72 °C. The PCR fragment was digested with
EcoRI and BamHI, ligated into pBluescript II SK,
and transferred from pBluescript II SK to a
NdeI/BamHI-digested pSP19 g10L vector. All
constructs were sequenced to exclude PCR errors.
Expression in E. coli
E. coli cells of strain C43(DE3) (18) were
transformed with the expression constructs and grown overnight in LB
medium supplemented with 100 µg ml Characterization of Enzyme Activity of Recombinant CYP71B6,
CYP71B7, and CYP83B1
The enzyme activity of CYP71B6, CYP71B7, and CYP83B1 was
measured in reaction mixtures in which spheroplasts of E. coli transformed with the respective expressing construct were
reconstituted with recombinant NADPH:cytochrome P450 reductase
(ATR1) from A. thaliana. In a typical enzyme assay, 25 µl
of spheroplasts and 0.06 units of NADPH:cytochrome P450
reductase were incubated in buffer containing 50 mM
Tricine, pH 8.1, 3 mM NADPH, 38 nCi (66 pmol) of
[U-14C]p-hydroxyphenylacetaldoxime, and 6 mM cysteine or N-acetylcysteine in a total
volume of 100 µl. Spheroplasts of E. coli C43(DE3) transformed with empty vector were used as controls. After incubation at 29 °C for 1 h, the reaction mixtures were extracted and
analyzed as described for the microsomal assays. Product formation from the oxime was linear with time within the first 10 min of incubation. For kinetic analysis, 5-50 µM
p-hydroxyphenylacetaldoxime was added to the standard
reaction mixture, which was incubated for 5 min and analyzed by TLC as
described above. Radiolabeled bands were visualized and quantified on a
STORM 840 PhosphorImager. For LC-MS analysis, 10 reactions of 100 µl
were done as described above except that radiolabeled
p-hydroxyphenylacetaldoxime was exchanged with 3 mM unlabeled oxime.
Substrate Binding Spectra
Substrate binding spectra were measured for recombinant CYP83B1
using partially purified enzyme. Purification was obtained by
temperature-induced Triton X-114 phase partitioning of E. coli spheroplasts as previously described (14). The substrate
binding spectra were performed on an SLM Aminco DW-2000 TM
spectrophotometer (SLM Instruments, Urbana, IL) at 12 °C using 10 µl of the Triton X-114-rich phase in 990 µl of 50 mM
KPi, pH 7.5, and 0.2 mM of either
p-hydroxyphenylacetaldoxime,
p-hydroxyphenylacetonitrile, 1-nitro-2-(p-hydroxyphenyl)ethane, or
p-hydroxyphenylacetamide.
Characterization of the Oxime-metabolizing Enzyme in
Microsomes from S. alba--
Biosynthetically active microsomes from
S. alba seedlings were isolated under conditions where
myrosinase was inhibited to prevent formation of inhibitory breakdown
products from the glucosinolates in the tissue (13). When
[U-14C]p-hydroxyphenylacetaldoxime and
cysteine were added to microsomes from S. alba in the
presence of NADPH, a cysteine-dependent radiolabeled compound accumulated in the reaction mixtures as evidenced by TLC
analysis (Fig. 1, column 2).
The compound was not detectable when boiled microsomes were used (Fig.
1, column 4), indicating that the reaction required the
presence of an active enzyme. The product formation was significantly
reduced when NADPH was not added to the reaction mixture, further
emphasizing that the reaction was enzyme-dependent and the
electron source was NADPH (Fig. 1, column 3). When the
reaction mixtures were extracted with ethyl acetate, the
cysteine-dependent compound remained in the water phase,
whereas the oxime accumulated in the ethyl acetate phase. The water
solubility indicated that the cysteine-dependent compound contained the polar groups of cysteine. The characteristics were consistent with the compound being the proposed intermediate
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, also referred to as cysteine conjugate. Such cysteine conjugates undergo cyclization with concomitant release of hydroxylamine to
produce the corresponding 2-substituted thiazoline-4-carboxylic acids
as evidenced by the chemically synthesized authentic standard S-(benzohydroximoyl)-L-cysteine (Fig.
2). The cyclization product (R)-2-(p-hydroxybenzyl)-thiazoline-4-carboxylic
acid formed from S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine
with [M + H]+ at m/z 238 was
identified by LC-MS (Fig. 3, A
and B). In the presence of tetcyclasis, an inhibitor of
cytochrome P450, the production of cysteine conjugate was significantly
reduced (Fig. 1, column 5). This indicated that the
oxime-metabolizing enzyme in the glucosinolate pathway is a cytochrome
P450-dependent monooxygenase.
When N-acetylcysteine was used as a sulfur donor in the
microsomal reaction mixtures containing
[U-14C]p-hydroxyphenylacetaldoxime and NADPH,
an N-acetylcysteine-dependent compound was
produced as evidenced on TLC (Fig. 1, column 7). The
N-acetylcysteine-dependent compound was not
present when boiled microsomes were used (Fig. 1, column 9),
and the product formation was significantly reduced when NADPH was not
added to the reaction mixture (Fig. 1, column 8), showing
that the reaction was enzyme-dependent and the electron
source was NADPH. The production was inhibited by tetcyclasis as was
seen for the cysteine conjugate compound (Fig. 1, column
10). The N-acetylcysteine-dependent
compound was identified by LC-MS analysis as
S-(p-hydroxyphenylacetohydroximoyl)-N-acetyl-L-cysteine, also referred to as N-acetylcysteine conjugate (Fig.
3, C and D). In contrast to the cysteine
conjugate, no cyclization of the N-acetylcysteine conjugate
took place, reflecting that the nucleophilic property of the amine
(cysteine) is considerably greater than that of the amide
(N-acetylcysteine). Rearrangement, as shown in Fig.
2D, can only take place if the N-acetyl group is
eliminated; this was not seen over the time course of the reaction. It
may take place, however, if the reaction is allowed to proceed for a
longer period of time during which hydrolysis occurs. By the LC-MS
analysis of the N-acetylcysteine conjugate two peaks were seen at retention times of 12.9 and 14.6 min corresponding to the
E- and Z-isomers of the hydroximino group
of the molecule, respectively. The two isomers had identical mass
spectra with [M + Na]+ at m/z 335, [M + H]+ at m/z 313, and fragments
at m/z 162, 130, and 107 (Fig. 3D). Very low sampling cone voltage was required for detection of the molecular ion. The conditions for detecting the molecular ion were
developed using the authentic standards
S-(benzohydroximoyl)-L-cysteine and
S-(benzohydroximoyl)-N-acetyl-L-cysteine.
When [U-14C]p-hydroxyphenylacetaldoxime and
NADPH were added to microsomes from S. alba in the absence
of a sulfur donor, a radiolabeled compound (X) accumulated in the
reaction mixtures as evidenced by TLC (Fig. 1, columns 1 and
6). The accumulation of this product (X) was reduced when a
sulfur donor was added (Fig. 1, columns 2 and 7).
Another compound accumulated in low amounts in ethyl acetate extracts
of the reaction mixtures (data not shown). The compound co-migrated
with authentic 1-nitro-2-(p-hydroxyphenyl)ethane. The
identity of the compound was confirmed by gas chromatography-MS (data
not shown). The specificity of the oxidation of oxime to nitro for the
glucosinolate biosynthetic pathway was investigated by testing the
ability of microsomes isolated from another glucosinolate-producing plant (T. majus) and a non-glucosinolate-producing plant
(Z. mays) to produce the nitro compound. Both T. majus and maize were able to convert the
p-hydroxyphenylacetaldoxime into
1-nitro-2-(p-hydroxyphenyl)ethane, showing that the reaction
is not specific for glucosinolate-producing plants.
Phylogenetic Analysis--
The activity of the oxime-metabolizing
enzyme in microsomes in S. alba was too low to pursue a
biochemical approach to identify the cytochrome P450 involved, which we
for evolutionary reasons expected to be related to the
oxime-metabolizing enzyme in the cyanogenic pathway. Considering the
rapid advance of the genome-sequencing project of A. thaliana, we took a bioinformatics approach to search for
homologues of CYP71E1 (P450ox), which presently is the only oxime-metabolizing enzyme identified in the biosynthetic pathway of
cyanogenic glucosides (12). In addition to the cytochromes designated
CYP71s, members of the CYP83 family belong correctly to the CYP71
family based on sequence
similarity.2 With the
sequences available at the given time (summer/autumn 1999), we combined
BLAST searches and sequence alignments, expression data (19), and
mutant phenotypes (20)3 to
identify candidates for the oxime-metabolizing enzyme in A. thaliana. Among the candidate genes found in the closest related subfamilies, CYP71B7, CYP71B6, and
CYP83B1 existed as full-length ESTs and were expressed in
E. coli. After completion of the genome-sequencing project
of A. thaliana, a phylogenetic tree of the CYP71 family was
made (Fig. 4).
Heterologous Expression of CYP71B6, CYP71B7, and CYP83B1 in E. coli--
CYP71B6, CYP71B7, and CYP83B1 were expressed in E. coli strain C43(DE3) by use of the expression vector pSP19 g10L.
CO difference spectrum with the characteristic peak at 450 nm was
obtained for CYP71B7 and CYP83B1 indicating the presence of functional
cytochrome P450 (Fig. 5). Based on the
peak at 450 nm, the expression levels of CYP71B7 and CYP83B1 were
estimated to be 50 and 430 nmol of cytochrome P450 (liter of
culture)
When radiolabeled oxime and NADPH were incubated with recombinant
CYP83B1 in the absence of a sulfur donor, a radiolabeled compound
accumulated in the reaction mixture that co-migrated with the
unidentified compound that accumulated in the S. alba microsomal reaction mixtures under the same conditions as evidenced by
TLC (Fig. 6, columns 3 and 8). Several attempts
to identify the compound (X) were unsuccessful. Accumulation of the
unidentified product (X) and of radiolabeling retained at the
application site was greatly reduced when a sulfur donor was added
(Fig. 6, columns 4 and 9). No detectable amounts
of 1-nitro-2-(p-hydroxyphenyl)ethane accumulated in the
reaction mixtures containing recombinant CYP83B1. Furthermore, neither
1-nitro-2-(p-hydroxyphenyl)ethane nor
p-hydroxyphenylacetonitrile was metabolized by CYP83B1 (data
not shown). This indicates that neither the nitro compound nor nitrile
is involved in the reaction catalyzed by CYP83B1.
Substrate Binding to CYP83B1--
We analyzed spectrally the
binding of substrate and potential intermediates to CYP83B1.
Recombinant CYP83B1 was partially purified from E. coli
spheroplasts by temperature-induced phase partitioning, which produced
a detergent rich-phase containing the majority of the cytochrome P450.
Recombinant CYP83B1 produced a type I spectrum with
p-hydroxyphenylacetaldoxime ( In the present study, we have characterized biochemically the
conversion of p-hydroxyphenylacetaldoxime to
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine in microsomes from S. alba and shown that the enzyme
activity was dependent on a cytochrome P450 monooxygenase. By combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified CYP83B1 as the oxime-metabolizing enzyme in
the glucosinolate pathway. We have used the tyrosine-derived oxime as
substrate, although A. thaliana does not produce the tyrosine-derived p-hydroxybenzylglucosinolate. We have,
however, previously shown that A. thaliana is capable of
converting p-hydroxyphenylacetaldoxime into
p-hydroxybenzylglucosinolate (22).
S-(Hydroximoyl)-L-cysteine is the most likely
S-alkyl thiohydroximate in the glucosinolate pathway for
several reasons. First, in vivo feeding studies have shown
that among other sulfur donors, cysteine is most efficiently
incorporated into glucosinolates (23). Second, the enzyme that converts
S-alkyl thiohydroximate to thiohydroximate is likely to be a
C-S lyase, and the characterized C-S lyases from plants hydrolyze
S-substituted cysteine derivatives and have an absolute
requirement for the presence of the For the oxime to be converted into the S-alkyl
thiohydroximate, it needs to be oxidized prior to conjugation with the
sulfur donor. We have shown the production of
1-nitro-2-(p-hydroxyphenyl)ethane from
p-hydroxyphenylacetaldoxime in microsomes from S. alba, T. majus, and Z. mays. Although
recombinant CYP83B1 is able to bind the nitro to the active site, no
nitro production or metabolism by the enzyme was detected, indicating
that nitro compounds are not intermediates in the glucosinolate
pathway. The form of the oxidized oxime might be the
corresponding aci-nitro compounds (8) or nitrile
oxides4 (Fig. 8), which are
interconvertible by a simple dehydration/hydration reaction and which
are very reactive compounds and subject to nucleophilic attack by
e.g. cysteine. Based on the
expected strong reactivity of the oxidized oxime, conjugation of the
sulfur donor is likely to be under strict control. The proposed
cysteine conjugation might be carried out by a glutathione
S-transferase. Alternatively, CYP83B1 may have a binding
site not only for the oxime but also for cysteine. In the absence of
added sulfur donor, an unidentified compound (X) accumulated in the
reaction mixture. This compound might be a furoxan (1,2,5-oxadiazole
2-oxide) formed by dimerization of nitrile oxide, or it might be a
conjugate derived from a nucleophile present in the reaction mixture.
Further studies are in progress to elucidate the mechanism for the
formation of the cysteine conjugate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-hydroxynitrile by
dehydrating the oxime to a nitrile, which is then C-hydroxylated to
form the
-hydroxynitrile (11, 12).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
N-Chlorosuccinimide
(280 g, 21 mmol) and HCl gas (40 ml) were added to benzaldoxime (2.42 g, 20.0 mmol) in dimethylformamide (40 ml) and stirred at room
temperature for 4 h. Water (150 ml) was added, and the reaction
mixture was extracted with diethyl ether (3 × 50 ml). The
combined ether phases were washed with water (2 × 50 ml), dried,
and concentrated in vacuo. The residue oil containing the
benzohydroximic acid chloride was dissolved in ethanol (50 ml) and
added to a solution of ice-cold L-cysteine (3.15 g, 20.0 mmol) in ethanol (100 ml). The mixture was treated with sodium
methoxide (sodium 0.74 g, 32.2 mmol; methanol 50 ml) and left
stirring for 3 h. The white precipitate was filtered, and the
filtrate was concentrated in vacuo, washed in water, and dried (3.42 g, 71%). NMR in D2O/NaOD: 1H
7.4-7.5 (5H, m, Ph), 3.28 (1H, dd, J 8.3, 3.9 Hz,
CH), 2.85 (1H, dd, J 13.2, 8.3 Hz,
CH2), 3.13 (1H, dd, J = 13.2, 3.9 Hz, CH2); 13C:
168.4 (COOH), 150.9 (S-C=N), 133.7, 128.4, 128.4, 129.1 (Ph), 53.3 (CH), 52.7 (CH2).
MS1: [M + Na]+
at m/z 263, [M + H]+ at
m/z 241, fragment ions at
m/z 224, m/z 195, m/z 154, and m/z 120. After
a few hours in methanol, the product underwent cyclization with
concomitant release of hydroxylamine to produce
(R)-2-phenyl-thiazoline-4-carboxylic acid. NMR
(Me2SO-d6): 1H
7.4-7.8 (5H, m, Ph), 5.17 (1H, t, J 9 Hz,
CH), 3.62 (2H, d, J 9 Hz,
CH2); 13C:
172.6 (S-C=N), 167.7 (COOH), 133.0 131.7 128.3 128.9 (Ph), 80.2 (CH), 35.7 (CH2). MS: [M + H]+ at m/z 208, fragment ion at
m/z 162.
Benzohydroximic
acid chloride (1.55 g, 10 mmol) diluted with tetrahydrofuran was
added dropwise to ice-cold N-acetyl-L-cysteine (1.63 g, 10 mmol) in tetrahydrofuran (30 ml) with triethylamine (4.04 g, 0.04 mmol). After stirring for an additional 30 min, dilute sulfuric
acid (5% v/v, 100 ml) was added, the tetrahydrofuran was removed
in vacuo, and the aqueous layer was left at room temperature for 48 h. The white precipitate was filtered, and the filtrate was
washed in diethyl ether and dried (2.06 g, 73%). NMR (D2O, NaOD): 1H: d 1.95 (3H, s,
CH3CO), 2.98 (1H, dd, J 5.0, 14.0, CHaHbCH), 3.00 (1H, dd, J
5.0, 14.0, CHaHbCH), 4.17 (1H, t,
J 5.0, CHaHbCH)
7.40-7.49 (5H, m, 5× ArH), 13C: d 24.8 (COCH3), 35.1 (CH2), 57.6 (CH), 131.4 (2× CH), 131.5 (CH),
131.6 (2× CH), 137.0 (Cq), 155.3 (C=NOH), 175.8 (CO), 179.2 (CO). MS:
[M + Na]+ at m/z 305, [M + H]+ at m/z 283, fragment ions at
m/z 208, m/z 162, and
m/z 130.
1 ampicillin. 2 ml of
culture was used to inoculate 200 ml of modified TB medium
containing 50 µg ml
1 ampicillin (14). The cultures were
grown at 37 °C at 250 rpm until A600 = 0.5-0.7. Then 1 mM thiamine and 75 µg ml
1
-aminolevulinic acid were added, and after an additional 1 h of
growth, the cultures were induced with 1 mM
isopropyl-
-D-thiogalactoside and subsequently grown at
28 °C for 40 h at 125 rpm. Spheroplasts were prepared as
previously described (14) except that glycerol was omitted in the final
buffer. The amount of expressed functional cytochrome P450 was
monitored by Fe2+·CO versus Fe2+
difference spectroscopy and quantified using an extinction coefficient of 91 mM
1 cm
1.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Production of the cysteine conjugate
(cys.cj.), the N-acetylcysteine
conjugate (N-Ac-cys.cj.), and an unidentified compound
(X) by S. alba microsomes.
[U-14C]p-Hydroxyphenylacetaldoxime was
incubated with microsomes from S. alba in the presence of
cysteine (columns 1-5) or N-acetylcysteine
(columns 6-10) as a sulfur donor. The reaction mixtures
were extracted with ethyl acetate, and the water phases were analyzed
by TLC. As a control boiled microsomes were used (columns
4 and 9). In the absence of a sulfur donor a
compound (X) accumulated (columns 1 and
6). The production of the conjugates was inhibited by the
cytochrome P450 inhibitor tetcyclasis (columns 5 and
10).
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Fig. 2.
LC-MS analysis of
S-(benzohydroximoyl)-L-cysteine.
A, LC-MS analysis of
S-(benzohydroximoyl)-L-cysteine (I)
dissolved in methanol. The total ion chromatogram shows the starting
material as Peak a and the pre-injection cyclized product,
(R)-2-phenyl-thiazoline-4-carboxylic acid (II) as
Peak b. The rising baseline between the two peaks stems from
on-column cyclization. B, mass spectrum of Peak
a: I gives rise to the ions [M + Na]+ at
m/z 263 and [M + H]+ at
m/z 241. C, mass spectrum of Peak b:
II gives rise to the ion [M + H]+ at
m/z 208. D, scheme illustrating the
cyclization process of
S-(benzohydroximoyl)-L-cysteine to
(R)-2-phenyl-thiazoline-4-carboxylic acid.
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Fig. 3.
Identification by LC-MS of
(R)-2-(p-hydroxybenzyl)-thiazoline-4-carboxylic
acid and of
S-(p-hydroxyphenylacetohydroximoyl)-N-acetyl-L-cysteine
produced by S. alba microsomes. A,
reconstructed ion chromatogram of m/z 238. B, mass spectrum of the cyclization product
(R)-2-(p-hydroxybenzyl)-thiazoline-4-carboxylic
acid of
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine
showing [M + H]+ at m/z 238 and
fragment ion at m/z 107. C,
reconstructed ion chromatogram (m/z 313) showing
the E- and Z-isomers of the product
S-(p-hydroxyphenylacetohydroximoyl)-N-acetyl-L-cysteine
(I) and reconstructed ion chromatogram
(m/z 152) showing E- and
Z-isomers of the substrate
p-hydroxyphenylacetaldoxime (II). D,
mass spectrum of
S-(p-hydroxyphenylacetohydroximoyl)-N-acetyl-L-cysteine
showing [M + Na]+ at m/z 335, [M + H]+ at m/z 313, and fragment ions at
m/z 162, 130, and 107.
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Fig. 4.
A phylogenetic tree of the CYP71 family in
A. thaliana and CYP71E1 (P450ox) from S. bicolor. The tree was constructed by alignment of the amino acid
sequences using ClustalW. Phylogenetic analysis was performed with the
Protdist and Fitch (Fitch-Margoliash and least squares method) programs
of the Phylip package. Despite their original name, the CYP83s belong
correctly to the CYP71 family. CYP76C4, CYP98A3, and CYP84A1 are
included as outgroups. CYP71B7, CYP71B6, and CYP83B1 were tested
earlier as candidates for the oxime-metabolizing enzyme in the
glucosinolate pathway.
1, respectively. The expression of CYP71B6 in
E. coli did not result in production of a protein giving a
peak at 450 nm in a CO difference spectrum; this, however, does not
exclude the presence of a functional protein (4). When
[U-14C]p-hydroxyphenylacetaldoxime and
cysteine or N-acetylcysteine were added to spheroplasts of
E. coli expressing CYP83B1 reconstituted with
NADPH:cytochrome P450 reductase in the presence of NADPH, a compound
accumulated in the reaction mixtures that co-migrated with the cysteine
conjugate (Fig. 6, column 4)
and the N-acetylcysteine conjugate (Fig. 6, column
9) produced by the S. alba microsomes as evidenced by
TLC. Formation of these products was furthermore shown to be dependent
on NADPH as the electron source (Fig. 6, columns 5 and
10). The products were not detected in reaction mixtures
containing E. coli spheroplasts harboring either the CYP71B6
or CYP71B7 (data not shown) or empty vector (Fig. 6, columns 1 and 6). The compounds produced from
p-hydroxyphenylacetaldoxime by recombinant CYP83B1 using
cysteine or N-acetylcysteine as a sulfur donor were
identified by LC-MS analysis as the cyclization product of
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine
or the (uncyclizing)
S-(p-hydroxyphenylacetohydroximoyl)-N-acetylcysteine (data not shown), respectively. Recombinant CYP83B1 has a pH optimum of
8.1, and kinetic studies showed that it has a turnover number of 16 min
1 and a Km of 68 µM
for p-hydroxyphenylacetaldoxime.
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Fig. 5.
Carbon monoxide difference spectra of CYP71B7
and CYP83B1. The Fe2+·CO versus
Fe2+ difference spectra were measured on spheroplasts of
E. coli expressing CYP71B7, CYP83B1, or empty vector. The
spectra were recorded at 12 °C.
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Fig. 6.
Production of the cysteine conjugate
(cys.cj.), the N-acetylcysteine
conjugate (N-Ac-cys.cj.), and an unidentified compound
(X) by CYP83B1 (83B1).
[U-14C]p-Hydroxyphenylacetaldoxime was
incubated with recombinant CYP83B1 reconstituted with NADPH:cytochrome
P450 reductase in the presence of cysteine (columns 2-5) or
N-acetylcysteine (columns 7-10) as a sulfur
donor. The reaction mixtures were extracted with ethyl acetate, and the
water phases were analyzed by TLC. In the absence of a sulfur donor a
compound (X) accumulated (columns 3 and
8). Empty vector was used as a control (c)
(columns 1 and 6).
max 390 nm and
min 427 nm), p-hydroxyphenylacetonitrile
(
max 391 nm and
min 427 nm), and
1-nitro-2-(p-hydroxyphenyl)ethane (
max 391 nm
and
min 427 nm) indicating a shift from a low to a high
spin state upon substrate binding (21) (Fig.
7, a, b, and
c). The amplitude increased in size upon incubation and
reached a stable maximum after ~30 min. No substrate binding spectrum
was obtained upon the addition of p-hydroxyphenylacetamide
(data not shown). Although neither the nitrile nor nitro compound was
metabolized by CYP83B1, the formation of type I binding spectra
demonstrated that CYP83B1 was able to bind these analogues.
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Fig. 7.
Substrate binding spectra obtained with
recombinant CYP83B1. Each cuvette contained 0.25 nmol of
recombinant CYP83B1 partly purified into a Triton X-114 detergent-rich
phase. 0.2 mM of substrate was added to the sample cuvette,
and spectra were recorded at 12 °C after 45 min.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydrogen atom and an
unsubstituted amino group in the cysteine moiety (10, 24). We have
succeeded for the first time in obtaining a mass spectrum providing
evidence for enzymatic synthesis of the proposed intermediate
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine.
In vitro
S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine
undergoes cyclization, which indicates that the proposed C-S
lyase is tightly coupled to the sulfur-conjugating enzyme in
vivo.
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Fig. 8.
The conversion of oxime to thiohydroximic
acid in the glucosinolate pathway. It is presently not known
whether the oxime is oxidized to an aci-nitro compound or a
nitrile oxide by CYP83B1.
Cyanogenic glucosides and glucosinolates are related groups of natural plant products derived from amino acids and with oximes as intermediates. Cyanogenic glucosides occur throughout the plant kingdom. This indicates that cyanogenesis arose as a very early evolutionary event. In contrast, glucosinolates are restricted to the order Capparales and the genus Drypetes in the distant order Euphorbiales (25). Cytochromes P450 belonging to the CYP79 family have been shown to catalyze the conversion of amino acids to oximes in both the cyanogenic and the glucosinolate pathway (3-7, 26), supporting the speculation that the biosynthesis of glucosinolates has evolved from the cyanogenic pathway (27).
If evolution of glucosinolates is based on a "cyanogenic
predisposition," this raises the question of how glucosinolates
evolved. In the biosynthetic pathway of the cyanogenic glucoside
dhurrin in S. bicolor, the oxime-metabolizing enzyme P450ox
(CYP71E1) converts the oxime to a -hydroxynitrile (26). Our working
hypothesis has been that a mutated homologue of P450ox,
P450mox, catalyzes the oxime-metabolizing step in the
biosynthetic pathway of glucosinolates (Fig.
9). According to the hypothesis,
P450mox would oxidize the oxime to a toxic or reactive
compound such as an aci-nitro or a nitrile oxide, which the
plant subsequently would have to detoxify. The post-oxime enzymes
include the proposed glutathione S-transferase and C-S lyase
in addition to the known glucosyltransferase and sulfotransferase.
These enzyme groups are known to be involved in general detoxification
reactions. This makes it likely that post-oxime enzymes have been
recruited from the detoxification processes but now are specialized for
glucosinolate production.
|
Several mutants of CYP83B1 have been reported (20, 28, 29). Recently, CYP83B1 was described as a regulator of auxin production by controlling the flux of indole-3-acetaldoxime into indole-3-acetic acid and indole glucosinolates (29). Characterization of recombinant CYP83B1 expressed in yeast showed that indole-3-acetaldoxime is a high affinity substrate for CYP83B1. The product obtained was shown to form adducts with a number of thiol compounds (29). It has been suggested that the glucosinolate pathway evolved from an indole-3-acetic acid biosynthetic pathway and not necessarily from the cyanogenic pathway (29). Cyanogenic glucosides are derived from only five protein amino acids (Tyr, Phe, Leu, Ile, and Val). The same few protein amino acids are also precursors for glucosinolates together with tryptophan, alanine, methionine, and chain-elongated derivatives of methionine and phenylalanine, which are not precursors of cyanogenic glucosides. This suggests that CYP79s of the glucosinolate pathway have acquired new substrate specificities after having diverged from the "cyanogenic" CYP79s. The biosynthesis of tryptophan-derived and chain-elongated amino acid-derived glucosinolates seems to be recent evolutionary events because indole glucosinolates are present in only four families in the order Capparales, namely in the Brassicaceae, Resedaceae, Tovaraceae, and Capparaceae (30), and because glucosinolates from chain-elongated amino acids are only found in Brassicaceae, Resedaceae, and Capparaceae (1). Considering the taxonomical distribution of cyanogenic glucosides and glucosinolates in the plant kingdom and of indole glucosinolates in only four families in the Capparales order (30), it appears likely that glucosinolates have evolved from a cyanogenic predisposition.
The CYP71 family is the largest cytochrome P450 family in A. thaliana with 47 members that cluster into different subgroups (Fig. 4). In the Arabidopsis genome, another member of the CYP83 family, CYP83A1, with 65% identity to CYP83B1 at the amino acid level has been identified. The high sequence similarity suggests that CYP83A1 may be an oxime-metabolizing enzyme in the glucosinolate pathway. The two CYP83s form a little subgroup that is located close to the CYP71E1, which is consistent with an evolutionary relationship between the oxime-metabolizing enzymes in the cyanogenic and the glucosinolate pathway.
Within the last few years considerable advances in our understanding of
glucosinolate biosynthesis have been achieved. Identification of the
oxime-metabolizing enzymes in both the cyanogenic and the glucosinolate
pathways combined with identification of substrate-specific oxime-producing CYP79s provides important molecular tools for metabolic
engineering of glucosinolate profiles and for introduction of
cyanogenic glucosides and other oxime-derived compounds in a
glucosinolate or a non-glucosinolate background.
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ACKNOWLEDGEMENTS |
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Emeritus Professor Anders Kjær, Professor Birger Lindberg Møller, and Dr. Mohammed Saddik Motawia are thanked for helpful discussions. Dr. Søren Bak is acknowledged for communicating the CYP83B1 knock-out phenotype.
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FOOTNOTES |
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* This work was financially supported by grants from The Royal Veterinary and Agricultural University (to C. H. H and L. D.), the Danish National Research Foundation, and the Director Ib Henriksens Foundation (to B. A. H.). IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Chemistry Dept., University of California, One Shields Ave., Davis, CA 95616.
¶¶ To whom correspondence should be addressed. Tel.: 45-35283342; Fax: 45-35283333; E-mail: bah@kvl.dk.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M102637200
4 A. Kjær, personal communication.
2 S. Paquette and S. Bak, personal communication.
3 S. Bak, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: MS, mass spectrometry; LC, liquid chromatography; EST, expressed sequence tag; PCR, polymerase chain reaction.
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REFERENCES |
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