 |
INTRODUCTION |
Glucosinolates are amino acid-derived secondary plant products
containing a sulfate and a thioglucose moiety. They are found in the
order Capparales, which includes the economically important oilseed
rape, Brassica vegetables, and the model plant
Arabidopsis thaliana L. Upon tissue damage, glucosinolates
are hydrolyzed by endogenous thioglucosidases (myrosinases) to produce
a variety of degradation products, typically isothiocyanates,
thiocyanates, and nitriles, which have a wide range of
biological effects (1, 2). Generally, the glucosinolate/myrosinase
system is believed to be involved in plant defense. In addition, it has
been shown that glucosinolates or rather the isothiocyanates,
particularly sulforaphane, the isothiocyanate of
4-methylsulfinylbutylglucosinolate, have anticarcinogenic properties
(3, 4). There is a rising interest in being able to control the level
of specific glucosinolates in crops to improve nutritional value and
pest resistance.
Glucosinolates are grouped into aliphatic, aromatic, and indole
glucosinolates, depending on whether they are derived from aliphatic
amino acids, phenylalanine and tyrosine, or tryptophan (for review, see
Ref. 5). The amino acid often undergoes a series of chain elongations
prior to entering the biosynthetic pathway, and the glucosinolate
product is often subject to secondary modifications such as
hydroxylations, methylations, and oxidations giving rise to the
structural diversity of glucosinolates. Biosynthetic intermediates
common to all glucosinolates are aldoximes, thiohydroximates, and
desulfoglucosinolates. The glucosinolates found in A. thaliana ecotype Columbia are derived from tryptophan, several
chain-elongated methionine homologues, chain-elongated phenylalanine
(6), and phenylalanine (7). However, the dihomomethionine-derived
glucosinolates 4-methylthiobutylglucosinolate and
4-methylsulfinylglucosinolate account for more than 50% of the total
glucosinolate content in the rosette leaves of A. thaliana
(8).
A key step in the biosynthesis of glucosinolates is the
N-hydroxylation of the precursor amino acids to the
corresponding aldoximes. In the biosynthesis of cyanogenic glucosides,
a group of natural plant products closely related to glucosinolates,
cytochromes P450 belonging to the CYP79 family catalyze the conversion
of amino acids to aldoximes (9-11). The nature of the enzymes
catalyzing the formation of aldoximes in glucosinolate biosynthesis has
been discussed controversially (12). Recently, evidence has been provided for the involvement of CYP79 homologues in the biosynthesis of
aromatic (7) and indole glucosinolates (13, 14). Regarding the
biosynthesis of aliphatic glucosinolates, extensive biochemical studies
with preparations of Brassica sp. and chain-elongated methionine homologues as substrates have suggested that aldoxime formation from these amino acids is not catalyzed by cytochromes P450,
but by flavin-dependent monooxygenases (15-17).
In the present paper, we report the identification of a cytochrome P450
of the CYP79 family, CYP79F1, which catalyzes the conversion of
dihomomethionine and trihomomethionine to 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. The reduced levels of
aliphatic glucosinolates and the accumulation of the chain-elongated precursor amino acids dihomomethionine and trihomomethionine in transgenic A. thaliana with CYP79F1 cosuppression is
consistent with the involvement of CYP79F1 in the biosynthesis of
aliphatic glucosinolates.
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EXPERIMENTAL PROCEDURES |
Plant Material--
A. thaliana ecotype Columbia was
used for all experiments. The plants were grown in a
controlled-environment Arabidopsis Chamber (Ar-60 I, Percival, Boone,
IA) at a photosynthetic flux of 100-200 µmol photons
m
2 s
1, 20 °C, and 70% humidity. Unless
otherwise stated, the photoperiod was 12-h light, 12-h dark.
Generation of the Construct for Escherichia coli
Expression--
The expression construct was derived from the
EST1 ATTS5112 (Arabidopsis
Biological Resource Center, Columbus, OH), which contains the
full-length sequence of CYP79F1. The CYP79F1
coding region was amplified from the EST by PCR with primer 1 (sense
direction; 5'-CTCTAGATTCGAACATATGGCTAGCTTTACAACATCATTACC) and primer 2 (antisense direction; 5'-CGGGATCCTTAAGGACGGAACTTTGGATA). Primer 1 introduces an XbaI site upstream of the start codon and an
NdeI restriction site at the start codon. To optimize the
construct for E. coli expression (18) primer 1 changes the
second codon from ATG to GCT and introduces a silent mutation in codon
5. Primer 2 introduces a BamHI restriction site immediately
after the stop codon. The PCR reaction was set up in a total volume of
50 µl in Pwo polymerase PCR buffer with 2 mM
MgSO4 using 2.5 units of Pwo polymerase (Roche Molecular Biochemicals), 0.1 µg of template DNA, 200 µM
dNTPs, and 50 pmol of each primer. After incubation of the reaction at 94 °C for 5 min, 20 PCR cycles of 15 s at 94 °C, 30 s
at 58 °C, and 2 min at 72 °C were run. The PCR fragment was
digested with XbaI and BamHI and ligated into an
XbaI/BamHI-digested pBluescript II SK
(Stratagene). The cDNA was sequenced to exclude PCR errors and
transferred from pBluescript II SK to an
NdeI/BamHI-digested pSP19g10L expression vector
(18).
DNA Sequencing and Computer Analysis--
Sequencing was
performed on an ALF-Express (Amersham Pharmacia Biotech) using a Thermo
Sequence Fluorescent-labeled primer cycle sequencing kit (7-deaza-dGTP)
(Amersham Pharmacia Biotech). Sequence computer analysis was done with
programs of the GCG Wisconsin Sequence Analysis Package. The GAP
program was used with a gap creation penalty of eight and a gap
extension penalty of two to compare pairs of sequences.
Expression in E. coli--
E. coli cells of strain
JM109 (Stratagene) and strain C43(DE3) (19) transformed with the
expression construct were grown overnight in LB medium supplemented
with 100 µg ml
1 ampicillin and used to inoculate 40 ml
of modified TB medium containing 50 µg ml
1 ampicillin,
1 mM thiamine, 75 µg ml
1
-aminolevulinic
acid, 1 µg ml
1 chloramphenicol, and 1 mM
isopropyl-
-D-thiogalactoside. The cultures were grown at
28 °C for 60 h at 125 rpm. The cells were pelleted and
resuspended in buffer composed of 0.2 M Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 M sucrose, and 0.5 mM
phenylmethylsulfonyl fluoride. Lysozyme was added to a final
concentration of 100 µg ml
1. After incubation for 30 min at 4 °C, Mg(OAc)2 was added to a final concentration
of 10 mM. Spheroplasts were pelleted, resuspended in 3.2 ml
of buffer composed of 10 mM Tris-HCl, pH 7.5, 14 mM Mg(OAc)2, and 60 mM KOAc, pH
7.4, and homogenized in a Potter-Elvehjem homogenizer. After DNase
treatment, glycerol was added to a final concentration of 30%.
Temperature-induced Triton X-114 phase partitioning resulted in the
formation of a detergent-rich phase containing the majority of the
cytochrome P450 and a detergent-poor phase (9). Functional expression
of CYP79F1 was monitored by Fe2+·CO versus
Fe2+ difference spectroscopy (20) performed on an SLM
Aminco DW-2000 TM spectrophotometer (SLM Instruments,
Urbana, IL) using 10 µl of Triton X-114-rich phase in 990 µl of
buffer containing 50 mM KPi, pH 7.5, 2 mM EDTA, 20% glycerol, 0.2% Triton X-100, and a few
grains of sodium dithionite. The amount of functional
CYP79F1 was estimated based on an absorption coefficient of
91 liters mmol
1 cm
1.
Measurements of Enzyme Activities--
The activity of CYP79F1
was measured in E. coli spheroplasts reconstituted with
NADPH:cytochrome P450 oxidoreductase purified from Sorghum
bicolor (L.) Moench as described earlier (21). In a typical enzyme
assay, 5 µl of spheroplasts and 4 µl of NADPH:cytochrome P450
reductase (equivalent to 0.04 units defined as 1 µmol of cytochrome
c/min) were incubated with substrate in buffer containing 30 mM KPi, pH 7.5, 3 mM NADPH, 3 mM reduced glutathione, 0.042% Tween 80, 1 mg
ml
1 L-
-dilauroylphosphatidylcholine in a
total volume of 30 µl. Reaction mixtures containing spheroplasts of
E. coli C43(DE3) transformed with empty vector were used as
controls in all assays. 3.3 µM
L-[U-14C]phenylalanine (453 mCi/mmol;
Amersham Pharmacia Biotech), 3.7 µM
L-[U-14C]tyrosine (449 mCi/mmol; Amersham
Pharmacia Biotech), 0.1 mM L-[methyl-14C]methionine (56 mCi/mmol; Amersham Pharmacia Biotech), and 24 µM-L-[side
chain-3-14C]tryptophan (56.5 mCi/mmol; PerkinElmer
Life Sciences) were tested as potential substrates. After incubation at
28 °C for 1 h, half of the reaction mixture was analyzed by TLC
on Silica Gel 60 F254 sheets (Merck) using toluene/ethyl
acetate 5:1 (v/v) as eluent. Radiolabeled bands were visualized and
quantified by STORM 840 PhosphorImager (Amersham Pharmacia Biotech).
For GC-MS analysis, 450 µl of reaction mixture containing 3.3 mM L-methionine (Sigma), 3.3 mM
DL-dihomomethionine or 3.3 mM
DL-trihomomethionine, respectively, were incubated for
4 h at 25 °C and extracted with a total volume of 600 µl
CHCl3. The organic phase was collected and evaporated, and
the residue was dissolved in 15 µl of CHCl3 and analyzed
by GC-MS. GC-MS analysis was performed on an HP5890 Series II gas chromatograph directly coupled to a Jeol JMS-AX505W mass spectrometer. An SGE column (BPX5, 25 m × 0.25 mm, 0.25-µm film thickness)
was used (head pressure, 100-kPa, splitless injection). The oven
temperature program was as follows: 80 °C for 3 min, 80 to 180 °C
at 5 °C/min, 180 to 300 °C at 20 °C/min, 300 °C for
10 min. The ion source was run in EI mode (70 eV) at 200 °C. The
retention times of the E- and Z-isomer of
authentic 5-methylthiopentanaldoxime were 14.3 and 14.8 min. The two
isomers had identical fragmentation patterns with
m/z 130, 129, 113, 82, 61, and 55 as the most
prominent peaks. The retention times of the E- and
Z-isomer of authentic 6-methylthiohexanaldoxime were 17.1 and 17.6 min. The two isomers had identical fragmentation patterns with
m/z 144, 143, 98, 96, 69, 61, and 55 as the most prominent peaks. DL-Dihomomethionine,
DL-trihomomethionine, 5-methylthiopentanaldoxime, and
6-methylthiohexanaldoxime were synthesized as described previously (22)
and authenticated by NMR spectroscopy.
For kinetic measurements of the conversion of
DL-dihomomethionine and DL-trihomomethionine to
the respective aldoximes by CYP79F1, enzymatic reactions were made
essentially as described above. 5 nmol of phenylacetaldoxime was added
to the reaction mixtures as internal standard. Product formation was
determined after 0, 5, 10, 20, 40, 70, and 120 min of incubation and
quantified based on authentic standards. The reaction mixtures were
extracted with a total volume of 600 µl of CHCl3. The
organic phase was collected and evaporated, and the residue was
dissolved in 15 µl of 50% ethanol and analyzed by LC-MS. LC-MS was
done on an HP1100 LC coupled to a Bruker Esquire-LC ion trap mass
spectrometer. The LC was performed on a C18 column
(Chrompack Inertsil 3 ODS-3 S15 × 3 COL CP 29126) using mixtures
of water (A) and acetonitrile (Fischer, "Far UV grade") (B) as
mobile phase at a flow rate of 0.3 ml min
1. The elution
program was as follows: 30% B (2 min), linear gradient 30-70% B (28 min), then linear gradient 70-100% (5 min). The electrospray ionization was done in positive ion mode. Each of the three aldoximes gave nicely separated peaks for the E- and
Z-forms. The 210-nm UV trace was detected by the diode array
detector and used for quantitation with the genuine software.
Generation of Transgenic Plants--
To construct plants, which
express the CYP79F1 cDNA under control of the CaMV35S
promoter (35S:CYP79F1 plants), the CYP79F1 cDNA was
PCR-amplified from the EST ATTS5112 (Arabidopsis Biological Resource
Center) using primer 3 (sense direction;
5'-AACTGCAGCATGATGAGCTTTACCACATC) and primer 4 (antisense direction;
5'-CGGGATCCTTAATGGTGGTGATGAGGACGGAACTTTGGATAA). Primer 3 is tailed with
a PstI restriction site. Primer 4 introduces four codons
coding for His before the stop codon and a BamHI restriction site after the stop codon. The PCR fragment containing the
CYP79F1 cDNA was digested with PstI and
BamHI, ligated into a
PstI/BamHI-digested pBluescript II SK and
sequenced to exclude PCR errors. The CYP79F1 cDNA was
placed under control of the CaMV35S promoter by ligation into a
PstI/BamHI-digested pSP48 (Danisco Biotechnology,
Denmark). The expression cassette was excised by XbaI
digestion and transferred to pPZP111 (23). Agrobacterium
tumefaciens strain C58 (24) transformed with this construct was
used for plant transformation by floral dip (25) using 0.005% Silwet
L-77 and 5% sucrose in 10 mM MgCl2. Seeds were
germinated on MS medium supplemented with 50 µg ml
1
kanamycin, 2% sucrose, and 0.9% agar. Transformants were selected after 2 weeks and transferred to soil. Seeds of these plants were harvested, and kanamycin-resistant T2 plants were selected
on the same medium. The procedure was repeated to obtain T3 plants.
HPLC Analysis of the Glucosinolate Content of Plant
Extracts--
The analysis was performed with tissue harvested from
9-week-old primary transformants and 7-week-old T3 plants.
Simultaneously grown wild-type plants of the same ages were used as
controls. The tissue (two to three rosette leaves from each plant) was
freeze-dried for 48 h. Glucosinolates were analyzed as
desulfoglucosinolates as follows: 3.5 ml of boiling 70% (v/v) methanol
were added to 20 mg of freeze-dried material, 75 µl of internal
standard (0.52 mM p-hydroxybenzylglucosinolate;
Bioraf, Denmark) were added, and the sample was incubated in a boiling
water bath for 4 min. Plant material was pelleted, and the pellet was
re-extracted with 3.5 ml of 70% (v/v) methanol and centrifuged. The
supernatants were pooled and analyzed by HPLC after sulfatase treatment
as described previously (7). The assignment of peaks was based on
retention times, and UV spectra were compared with standard compounds.
Glucosinolates were quantified in relation to the internal standard and
by use of response factors (8, 26).
Analysis of the Amino Acid Content of Plant
Extracts--
Rosette leaves of 7-week-old plants (250 mg from each
plant) were frozen in liquid nitrogen and homogenized using mortar and pestle. The tissue was extracted in 2.5 ml of 80% methanol. The plant
material was pelleted (20,000 × g, 10 min) and
re-extracted in 2.5 ml of 80% methanol. The methanol phases were
combined and dried in vacuo, and the residue was dissolved
in 100 µl of water. The individual protein amino acids in the sample
were identified and quantified on an Ultropac 8 Resin reverse phase
HPLC column (200 × 4.6 mm) on a Biochrom 20 amino acid analyzer
(Amersham Pharmacia Biotech) according to the manufacturer. The elution program was modified to identify and quantify the chain-elongated homologues of methionine, dihomomethionine, and trihomomethionine. Each
plant was analyzed in triplicate.
For quantification of dihomomethionine in the plant material, the
sample was subjected to two elution programs. Program 1 was as follows:
53 °C for 7 min, buffer A; 50 °C for 35 min, buffer A; 95 °C
for 34 min, buffer A. Program 2 was as follows: 53 °C for 7 min,
buffer A; 58 °C for 12 min, buffer B; 95 °C for 25 min, buffer C. Buffer A was 0.2 M sodium citrate, pH 3.25, buffer B was
0.2 M sodium citrate, pH 4.25, and buffer C was 1.2 M sodium citrate, pH 6.25. In program 1, phenylalanine
and dihomomethionine coeluted at 63.6 min. In program 2, tyrosine and
dihomomethionine coeluted at 25.3 min. Dihomomethionine was quantified
as the difference between the peak area corresponding to phenylalanine
and dihomomethionine in program 1 and the peak area corresponding to
phenylalanine in program 2, and as the difference between the peak area
corresponding to tyrosine and dihomomethionine in program 2 and the
peak area corresponding to tyrosine in program 1. The response factor
for dihomomethionine was determined using an authentic standard.
For quantification of trihomomethionine in the plant material, the
sample was subjected to program 3, which was as follows: 53 °C for 7 min, buffer A; 58 °C for 5 min, buffer B; 95 °C for 7 min, buffer
B; 95 °C for 25 min, buffer C. Trihomomethionine eluted at 29.0 min
and was quantified as the peak area using a response factor determined
with an authentic standard.
Synthesis of Control RNA--
RNA was synthesized from
pBluescript II SK vector (Stratagene) linearized by digestion with
ScaI. The synthesis reaction was set up in a total volume of
100 µl in Transcription Optimized buffer (Promega) supplemented with
500 µM rNTPs, 10 mM dithiothreitol, 100 units
of RNasin ribonuclease inhibitor (Promega), 3 µg of linearized
pBluescript II SK, and 50 units of T3 RNA polymerase (Promega). After
incubation at 37 °C for 2 h, 20 units of RNase-free DNase was
added, and the reaction was incubated at 37 °C for another 1 h.
Following phenol-CHCl3 extraction and precipitation with ethanol (27), the RNA was dissolved in diethylpyrocarbonate-treated water. The control RNA was used to check for inhibition of RT reactions
by components of RNA preparations obtained from different plant tissues.
Expression Analysis by RT-PCR--
To study the expression
pattern of CYP79F1 in wild-type A. thaliana, the following
tissues were investigated: 1) total plant tissue of 4-week-old plants
(grown in 8-h light/16-h dark); 2) rosette leaves (without petioles)
and 3) above-ground parts of 5-week-old plants (before onset of floral
transition; grown at 8-h light/16-h dark); 4) rosette leaves (without
petioles); and 5) cauline leaves of flowering plants (9 weeks old;
grown at 12-h light/12-h dark to induce flowering). To study transcript
levels in 35S:CYP79F1 plants, rosette leaves of 7-week-old 35S:CYP79F1 plants and of simultaneously grown 7-week-old wild-type plants were analyzed.
Total RNA was isolated from plant tissue using TRIzol reagent (Life
Technologies, Inc.). The RNA was quantified spectrophotometrically and
used to synthesize first-strand cDNA. To ensure linearity of the
RT-PCR, first-strand cDNA synthesis was performed on 1, 0.3, and
0.1 µg of each pool of RNA. The cDNA was synthesized in First
Strand buffer (Life Technologies, Inc.) supplemented with 0.5 mM dNTPs, 10 mM dithiothreitol, 200 ng of
random hexamers (Amersham Pharmacia Biotech), 3 pg of control RNA
(internal standard), and 200 units of Superscript II reverse
transcriptase (Life Technologies, Inc.) in a total volume of 20 µl.
The reaction mixture was incubated at 27 °C for 10 min followed by
incubation at 42 °C for 50 min and inactivation at 95 °C for 5 min. The RT reactions were purified by means of a PCR purification kit
(Qiagen; elution with 50 µl of 1 mM Tris buffer, pH 8). 2 µl of the purified RT reactions was subjected to PCR. The PCR
reactions were set up in a total volume of 50 µl in PCR buffer (Life
Technologies, Inc.) supplemented with 200 µM dNTPs, 1.5 mM MgCl2, 50 pmol of sense primer, 50 pmol of
antisense primer, and 2.5 units of Platinum Taq DNA
polymerase (Life Technologies, Inc.). The PCR program was as follows: 2 min at 94 °C, 23-37 cycles (depending on transcript as specified
below) of 30 s at 94 °C, 30 s at 53-57 °C (depending
on transcript as specified below), 50 s at 72 °C. The following
primers and specific conditions were used: for CYP79F1
primer 5 (sense direction; 5'-AAAGCTCAATGCGTAGAAT) and primer 6 (antisense direction; 5'-TTTTTAGACACCATCTTGTTTTCTTCTTC) with 53 °C
and 32 cycles, for CYP79F2 primer 7 (sense direction; 5'-AAAGCTCAATGCGTCGAAT) and primer 8 (antisense direction;
5'-GCGTCGAAACACATCACAGAG) with 53 °C and 37 cycles, for
CYP79B2 primer 9 (sense direction; 5'-AGACGAACAAGGCAACCCA)
and primer 10 (antisense direction; 5'-TCATAAAATATATACGGCGTCG) with
53 °C and 34 cycles, for CYP79B3 primer 11 (sense
direction; 5'-GGACGAAGCTGGCCAGCCT) and primer 12 (antisense direction;
5'-TCCTCGCCGTACGTCACCG) with 55 °C and 32 cycles, for
actin1 primer 13 (sense direction; 5'-TGGAACTGGAATGGTTAAGGCTGG) and primer 14 (antisense direction; 5'-TCTCCAGAGTCGAGCACAATACCG) with 57 °C and 23 cycles, for control RNA (internal standard) primer 15 (sense direction;
5'-TGTAGCGGCGCATTAAGC) and primer 16 (antisense direction;
5'-CAAAAGAATAGACCGAGATAGGG) with 57 °C and 32 cycles. 10 µl of the
PCR reactions were analyzed by gel electrophoresis on 1% agarose gels.
Bands were visualized by ethidium bromide staining and quantified on a
Gel Doc 2000 Transilluminator (Bio-Rad). Primers and PCR conditions
were optimized to ensure amplification of the specific cDNAs. No
PCR products from genomic DNA were obtained. PCR analysis of the
internal standard showed that the RT reactions ran with the same
efficiency in samples prepared with different amounts of RNA isolated
from different plant tissues.
 |
RESULTS |
Expression of CYP79F1 in E. coli and Identification of
Substrates--
Cytochromes P450 of the CYP79 family have previously
been shown to be involved in the biosynthesis of cyanogenic glucosides and glucosinolates. CYP79F1 is one of several
CYP79 homologues identified in the genome of A. thaliana. The deduced amino acid sequence of CYP79F1
has 88% identity with the deduced amino acid sequence of
CYP79F2 and 39-46% identity with other CYP79
homologues from glucosinolate and cyanogenic glucoside-containing
species. A full-length EST of CYP79F1 (ATTS5112) was
identified by a data base search. The cDNA obtained from the EST
clone was used for expression of CYP79F1 in E. coli using
the vector pSP19g10L, which is optimized for expression of cytochromes
P450 (18). The CYP79F1 expression construct was transformed into two
different E. coli strains, C43(DE3) and JM109. A CO
difference spectrum with the characteristic peak at 450 nm was obtained
for CYP79F1 expressed in strain C43(DE3), indicating the presence of
functional cytochrome P450 (Fig. 1).
Based on the peak at 450 nm, the expression level of CYP79F1 in
E. coli C43(DE3) was estimated to be 110 nmol of cytochrome
P450 (liters of culture)
1. A peak at 418 nm was
detected in all preparations independent of whether E. coli
was transformed with a cytochrome P450 expression construct or the
empty vector. The origin of the peak at 418 nm is unknown, but could
possibly be derived from endogenous heme proteins (28). The absence of
a peak at 450 nm in the CO difference spectrum obtained with a
preparation of E. coli strain JM109 harboring the CYP79F1
expression construct indicates low expression level or failure of
expression of functional protein. Recombinant CYP79F1 was therefore
obtained by use of strain C43(DE3) for all further experiments.

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Fig. 1.
Fe2+·CO versus
Fe2+ difference spectrum of CYP79F1 expressed in
E. coli. Spectra were recorded of the
detergent-rich phase from temperature-induced Triton X-114 phase
partitioning of spheroplasts of E. coli transformed with the
expression construct for CYP79F1 (A) or empty vector
(B).
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To identify substrates of CYP79F1, activity measurements were carried
out using spheroplasts of E. coli C43(DE3) reconstituted with NADPH:cytochrome P450 reductase from S. bicolor. When
the reaction mixture containing CYP79F1 was incubated with
DL-dihomomethionine, two compounds, which were not present
in the control reactions, were detected by GC-MS (Fig.
2). The retention times and the mass spectral fragmentation patterns of these compounds were identical with
those of the E/Z-isomers of the authentic
standard of 5-methylthiopentanaldoxime. When
DL-trihomomethionine was administered to the reaction
mixture containing CYP79F1, two compounds with retention times and
fragmentation pattern identical to those of the
E/Z-isomers of the authentic standard of
6-methylthiohexanaldoxime were detected by GC-MS. The formation of both
5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime was linear
with time within 40 min. Product formation combined with cytochrome
P450 quantification allow the estimation of turnover number of
0.23 ± 0.01 min
1 for dihomomethionine and 0.15 ± 0.01 min
1 for trihomomethionine. No aldoximes were
produced using boiled enzyme preparation. Administration of
L-methionine, L-phenylalanine, L-tyrosine, and L-tryptophan to the reaction
mixtures containing recombinant CYP79F1 did not result in the formation
of detectable amounts of the corresponding aldoximes.

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Fig. 2.
GC-MS analysis of 5-methylthiopentanaldoxime
and 6-methylthiohexanaldoxime produced by CYP79F1 from dihomomethionine
and trihomomethionine, respectively. Dihomomethionine
(A-C) and trihomomethionine (D-F) were
administered to spheroplasts of E. coli harboring CYP79F1
(A, D) or empty vector (B,
E) in the presence of NADPH:cytochrome P450 reductase from
S. bicolor. The samples were incubated at 25 °C for
4 h and extracted with chloroform, and the organic phase was
analyzed by GC-MS. A and B, ion trace at
m/z 130. C, mass spectrum of the
5-methylthiopentanaldoxime with a retention time of 14.3 min from
A. D and E, Ion trace at
m/z 144. F, mass spectrum of the
6-methylthiohexanaldoxime with a retention time of 17.1 min from
D.
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Analysis of 35S:CYP79F1 Plants--
We have produced transgenic
A. thaliana expressing the CYP79F1 cDNA under
the control of the CaMV35S promoter to study the effect of altered
expression levels of CYP79F1 on the content and composition of
glucosinolates. Nine independent primary 35S:CYP79F1 transformants were
investigated, and four were selected for analysis through the following
two generations. The four primary transformants had dramatically
reduced levels of short-chain aliphatic glucosinolates (plants S7 and
S9) or slightly increased levels of these glucosinolates (plants S3 and
S5)(Fig. 3). The four plants had a
morphological phenotype characterized by reduced growth rates, reduced
apical dominance, and production of multiple axillary shoots at the
time of floral transition resulting in bushy plants. Analysis of plants of the T2 and T3 generations showed that the
observed phenotype of changed glucosinolate profile and bushy
appearance was not stable. However, T3 plants of the
primary transformant S3 (S3.8.1-S3.8.4) had the characteristic bushy
phenotype (Fig. 4) and a dramatically reduced content of aliphatic glucosinolates (Table
I). The effect was very pronounced for
the glucosinolates derived from short-chain methionine homologues,
i.e. homomethionine and dihomomethionine. The levels of
3-methylsulfinylpropylglucosinolate (derived from homomethionine),
4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate (both derived from dihomomethionine) were reduced to 9-13% compared with wild-type. The level of glucosinolates derived from methionine elongated by three to six methylene groups was reduced to about 30 to
50% compared with wild-type. The total content of indole glucosinolates was increased to about the double of the level in
wild-type plants. Plants S3.8.2 and S3.8.3 had the most pronounced phenotype and were selected for analysis of their content of the biochemically identified substrates of CYP79F1, dihomomethionine and
trihomomethionine (Fig. 5). Both plants
accumulated high amounts of the CYP79F1 substrates. Plant S3.8.2
contained as much as 50 times more dihomomethionine and ten times more
trihomomethionine than wild-type plants. RT-PCR analysis of plant
S3.8.4 showed that the levels of CYP79F1 and
CYP79F2 transcripts were strongly reduced compared with the
level in wild-type plants, suggesting that introduction of the
transgene had led to cosuppression (Fig. 6). The transcript level of
CYP79B2 was slightly increased compared with the level in
wild-type plants (Fig. 6).

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Fig. 3.
Major glucosinolates in 35S:CYP79F1 primary
transformants. Rosette leaves of 35S:CYP79F1 plants and three
wild-type plants were analyzed by HPLC. The value for wild-type
represents the mean (± S.E.) of three individual plants.
3msp, 3-methylsulfinylpropylglucosinolate; 4mtb,
4-methylthiobutylglucosinolate; 4msb,
4-methylsulfinylglucosinolate; 8mso,
8-methylsulfinyloctylglucosinolate; i3m,
indol-3-ylmethylglucosinolate; 1moi,
1-methoxyindol-3-ylglucosinolate; 4moi,
4-methoxyindol-3-ylglucosinolate.
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Fig. 4.
Morphological phenotype of 35S:CYP79F1
plants. A, 7-week-old 35S:CYP79F1 plant (S3.8.2)
(right), wild-type plant of the same age (left).
B, 11-week-old 35S:CYP79F1 plant (S3.8.2)
(right), wild-type plant of the same age
(left).
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Table I
Glucosinolate content of 35S:CYP79F1 T3 plants
The glucosinolate content of rosette leaves of plants S3.8.1-S3.8.4
and four wild-type plants was determined by HPLC as described under
"Experimental Procedures." Values are given as mean (±S.E.)
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Fig. 5.
Accumulation of chain-elongated methionine
homologues in rosette leaves of 35S:CYP79F1 plants and wild-type
plants. Extracts from two T3 plants derived from the
primary transformant S3 and two wild-type plants were analyzed by HPLC.
Each bar represents the mean (± S.E.) of four measurements
on one individual plant.
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Fig. 6.
RT-PCR analysis of transcript levels of CYP79
homologues in rosette leaves of a 35S:CYP79F1 plant (S3.8.4). RT
reactions were performed on 1, 0.3, and 0.1 µg of plant RNA. The
experiment has been performed three times with similar results.
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S3.8.1-S3.8.4 had normal growth rates, but the edges of the leaves
were curling upwards (Fig. 4). Before floral transition became
apparent, reduced apical dominance resulted in production of multiple
axillary shoots, which later developed into lateral influorescences.
The plants had reduced fertility and produced only a few normal
siliques and many short siliques with no or only few seeds.
CYP79F1 Expression Analysis--
The level of CYP79F1
transcript was investigated in rosette leaves of plants of different
developmental stages and in cauline leaves (Fig.
7). The transcript was detected in all
tissues examined. The transcript level increased with maturation of the
plants. The expression level was approximately four times higher in
rosette leaves of 9-week-old flowering plants than in rosette leaves of 5-week-old plants. When the above-ground parts of 5-week-old plants were analyzed, less CYP79F1 transcript was detected than
when only rosette leaves of the same plants were analyzed. This
indicates that CYP79F1 is expressed at higher levels in rosette leaves
than in petioles.

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Fig. 7.
Expression analysis of CYP79F1
in A. thaliana. A, RT-PCR
analysis with CYP79F1-specific primers. RT reactions were
performed on 1, 0.3, and 0.1 µg of plant RNA. B, control
for equal template loading in the RT reactions. 1 µg of RNA from each
tissue was analyzed by agarose gel electrophoresis. 1, total
plant tissue of 4-week-old plants; 2, rosette leaves
(without petioles) of 5-week-old plants; 3, above-ground
parts of 5-week-old plants; 4, rosette leaves (without
petioles) of 9-week-old flowering plants; 5, cauline leaves
of 9-week-old flowering plants.
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DISCUSSION |
The CYP79 family comprises multifunctional cytochromes P450 that
catalyze two consecutive N-hydroxylations of amino acids followed by dehydration and decarboxylation resulting in the formation of aldoximes (9). CYP79F1 is one of several CYP79
homologues identified in the genome of A. thaliana
(available on the Web). In the present paper we report that
CYP79F1 is an N-hydroxylase catalyzing the conversion of
dihomomethionine and trihomomethionine to 5-methylthiopentanaldoxime
and 6-methylthiohexanaldoxime, respectively, a key step in the
biosynthesis of aliphatic glucosinolates. CYP79F1 is the first
aldoxime-forming enzyme in the biosynthesis of aliphatic glucosinolates
to be heterologously expressed and characterized.
Using an EST clone containing the CYP79F1 cDNA we have expressed
CYP79F1 in E. coli. The recombinant protein has the spectral characteristics of a cytochrome P450 enzyme. Both dihomomethionine and
trihomomethionine are metabolized by CYP79F1 resulting in the formation
of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime as proven
by GC-MS analysis and comparison with authentic standards. Neither
methionine nor the other protein amino acids tested are substrates of
CYP79F1. Thus CYP79F1 seems to convert specifically chain-elongated
methionine homologues to the corresponding aldoximes. Previously,
aliphatic amino acids with different chain lengths have been shown to
be substrates of the same CYP79 as demonstrated for CYP79D1 and CYP79D2
from cassava (Manihot esculenta Crantz), which converts both
valine and isoleucine to the corresponding aldoximes in the
biosynthesis of the cyanogenic glucosides linamarin and lotaustralin
(10). In contrast, CYP79A2 from A. thaliana has been shown
to convert specifically phenylalanine, but not homophenylalanine, to
the corresponding aldoxime (7).
Heterologous expression in E. coli of CYP79F1 was
accomplished in the strain C43(DE3). Strain C43(DE3) is a mutant
E. coli strain, which has been selected for its ability to
accommodate high levels of heterologous membrane proteins (19).
C43(DE3) has been used successfully for cytochrome P450 expression
(14). In the present study, the use of strain C43(DE3) enabled
sufficient expression levels for spectral characterization of CYP79F1,
whereas this could not be accomplished in the strain JM109.
Two lines of evidence for the involvement of CYP79F1 in glucosinolate
biosynthesis are provided by the analysis of transgenic A. thaliana containing the CYP79F1 cDNA under control
of the CaMV35S promoter. First, several independent 35S:CYP79F1
transformants have either reduced or increased levels of aliphatic
glucosinolates, and reduced CYP79F1 transcript levels in
plants of the T3 generation are accompanied by a
dramatically reduced content of aliphatic glucosinolates. Second, the
substrates dihomomethionine and trihomomethionine of CYP79F1 accumulate
in the plants with reduced content of aliphatic glucosinolates as would
be expected upon down-regulation of CYP79F1. The accumulation of the
chain-elongated methionines indicates that the enzymes catalyzing the
chain elongation of methionine (29) are not subject to feed-back
inhibition by the chain-elongated product. Furthermore, it suggests
that the enzymes that catalyze additional chain elongation cycles are
rate-limiting in the biosynthesis of longer chain methionine homologues.
Comparison of the biochemical data for recombinant CYP79F1 and the
glucosinolate profiles of the 35S:CYP79F1 plants raises the question
whether CYP79F1 metabolizes not only dihomo- and trihomomethionine, but
also other chain-elongated methionine homologues produced in A. thaliana. Although the content of all aliphatic glucosinolates is
reduced in the transgenic plants compared with wild-type, the effect is
most pronounced for the glucosinolates derived from homo- and
dihomomethionine. The decrease in the levels of other aliphatic
glucosinolates than the dihomo- and trihomomethionine-derived ones
might be explained by a broad substrate specificity of CYP79F1 for
chain-elongated methionine homologues or by cosuppression not only of
the CYP79F1 transcript but also of transcripts of other
CYP79 homologues involved in the biosynthesis of aliphatic glucosinolates. As demonstrated by RT-PCR, introduction of the transgene resulted in down-regulation of both CYP79F1 and
CYP79F2. CYP79F1 is 88% identical at the amino acid level
to CYP79F2, and the two genes are located on the same
chromosome, only separated by 1638 bp. This suggests that the two genes
have been formed by gene duplication and that they might catalyze
similar reactions. Because further investigations of the substrate
specificity of CYP79F1 are limited by the unavailability of substrates,
isolation of A. thaliana knock-out mutants of CYP79
homologues may facilitate such investigations. From the results of the
RT-PCR, it appears that CYP79B2 is up-regulated
approximately 2-fold in the 35S:CYP79F1 plants, resulting in an
increased content of indole glucosinolates.
The transgenic A. thaliana plants with altered content of
aliphatic glucosinolates possess a characteristic morphological phenotype characterized by production of multiple axillary shoots. A. thaliana has been reported to be able to tolerate
overexpression of cytochromes P450 of the CYP79 family leading to a 2- to 5-fold increase in glucosinolate content (7, 30) without similar changes in the appearance of the plants. Therefore it seems unlikely that the morphological changes result from the presence or absence of
specific glucosinolates. The accumulation of very high levels of
chain-elongated methionine homologues in the transgenic plants suggests
that the morphological phenotype may be a pleiotropic effect caused by
disturbance of the plant's sulfur metabolism, in which methionine
plays a central role. Repression of cystathionine-
-synthase, a key
enzyme in methionine biosynthesis, results in plants characterized by
formation of a cluster of apical shoots at the time of floral transition and inability to produce flowers (31). 80% of the methionine synthesized by a plant is incorporated into
S-adenosyl-methionine (32), which plays a central role in
many biosynthetic processes, e.g. methylation reactions such
as cytosine methyltransferase-catalyzed DNA methylation (33). One of
the morphological changes seen in cytosine methyltransferase antisense
plants is production of multiple axillary shoots (34). The onset of the
morphological changes in CYP79F1-cosuppressed plants at the time of
floral transition may be due to the requirement for methionine to
support flower development. Alternatively, it coincides with an
increase in the level of CYP79F1 expression in wild-type plants. The
relation between CYP79F1 down-regulation and the morphological
phenotype is the subject of future investigations.
Based on biochemical studies using microsomal enzyme preparations
from species of the Brassicaceae, it has previously been proposed that
the conversion of dihomo-, trihomo-, and tetrahomomethionine to their
corresponding aldoximes is catalyzed by flavin-containing monooxygenases and not by cytochromes P450 (15-17). However, the conversion of chain-elongated methionine derivatives by
flavin-containing monooxygenases was measured by indirect enzyme assays
(15), in which the release of CO2 was used as a measure for
enzyme activity. Despite high enzyme activity, the corresponding
aldoximes have never been documented in these assays. Our study
provides unequivocal evidence that a cytochrome P450 of the CYP79
family, CYP79F1, catalyzes the aldoxime formation in the biosynthesis
of the dihomo- and trihomomethionine-derived glucosinolates in A. thaliana.
Glucosinolates are related to cyanogenic glucosides, because both
groups of natural products are derived from amino acids and have
aldoximes as intermediates. Because cyanogenic glucosides are found
throughout the plant kingdom and the occurrence of glucosinolates is
limited to the order Capparales and the genus Drypetes in
the order Euphorbiales, it has been hypothesized that glucosinolates have evolved from cyanogenic glucosides and that homologous enzymes catalyze the common aldoxime-forming step (35, 36). Although biochemical data have indicated that other enzyme systems may catalyze
this reaction (12), our data provide the final evidence that CYP79
homologues catalyze not only the conversion of aromatic amino acids and
tryptophan to their corresponding aldoximes but also the conversion of
aliphatic amino acids. This strongly indicates that the evolution of
glucosinolates is based on a "cyanogenic predisposition" (35).
Furthermore, it suggests that the CYP79 homologues in glucosinolate
biosynthesis developed new substrate specificities (e.g.
toward tryptophan and chain-elongated methionines) after having
diverged away from the CYP79 homologues involved in the biosynthesis of
cyanogenic glucosides.
Degradation of 4-methylsulfinylbutylglucosinolate by myrosinase in
broccoli sprouts leads to the formation of the isothiocyanate product
sulforaphane. As demonstrated in rats, extracts of broccoli sprouts
have a pronounced protective effect against breast cancer (4), and
sulforaphane has been identified as the principle active agent
(3). The identification of a gene involved in the biosynthesis of
4-methylsulfinylbutylglucosinolate is an important step in the
development of functional foods that release elevated levels of
sulforaphane. 5-Methylthiopentanaldoxime is not only the precursor of
4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate but also the precursor of a number of glucosinolates with secondary modifications of the side chain. Besides their occurrence in A. thaliana, such glucosinolates are important constituents of
Brassica crops and vegetables. For example, the major
glucosinolate in B. napus, the goitrogenic
2-hydroxy-3-butenylglucosinolate, is formed by side-chain modification
of 4-methylthiobutylglucosinolate (37). The occurrence of
2-hydroxy-3-butenylglucosinolate in B. napus restricts the
use of the protein-rich seed cake as animal feed. Thus availability of
biosynthetic genes has great potential for the development of crops
with reduced levels of such toxic glucosinolates while retaining
glucosinolates with desirable effects, e.g. for pest resistance.
In conclusion, we have shown that CYP79F1 catalyzes the conversion of
dihomomethionine and trihomomethionine to 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively, in the biosynthesis of
aliphatic glucosinolates in A. thaliana. The identification of CYP79F1 provides an important tool for tissue-specific alterations of the level of aliphatic glucosinolates to improve the nutritional value of crop plants and vegetables as well as pest resistance. In
addition, the availability of a biosynthetic gene for aliphatic glucosinolates is a valuable means for studying the physiological role
of these glucosinolates in plants, e.g. in plant-insect interactions.