From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
Received for publication, September 20, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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Matairesinol is a central precursor in
planta in the biosynthesis of numerous lignans, including that of
the important antiviral and anticancer agent, podophyllotoxin. In
this study, the ~32-kDa NAD-dependent
secoisolariciresinol dehydrogenase, which catalyzes the
enantiospecific conversion of ( The lignans are a structurally diverse class of vascular plant
metabolites with a broad range of medicinal/health protective roles in
addition to important physiological functions in planta (1).
For example, podophyllotoxin 1 has antiviral properties, and
etoposide 2 and teniposide 3 derivatives (2) (see
Fig. 1) are representatives of only a
handful of plant compounds extensively employed in cancer treatment; in
addition, the structurally related trachelogenin 4 possesses
anti-HIV properties (3). Furthermore, matairesinol 5 and
secoisolariciresinol 6 confer dietary protection to humans,
particularly against the onset of breast and prostate cancers (4). Both
compounds 5 and 6 are present to different
extents in various whole-grain cereal foods, seeds and berries, and are
converted by intestinal microflora (5) during digestion to form the
mammalian lignans, enterolactone 7 and enterodiol
8 (Fig. 1); the latter two compounds are considered as being
specifically responsible for the observed reductions in these
malignancies (6).
)-secoisolariciresinol into (
)-matairesinol in Forsythia intermedia, was purified
>6,000-fold to apparent homogeneity. The 831-base pair cDNA clone
encoding this 277-amino acid protein was next obtained from a
library constructed from F. intermedia stem tissue, whose
fully functional recombinant protein, produced by expression of this
cDNA in Escherichia coli, catalyzed the same
enantiospecific conversion via the corresponding lactol intermediate. A
homologous secoisolariciresinol dehydrogenase gene was also isolated
from a Podophyllum peltatum rhizome cDNA library, whose
834-base pair cDNA clone encoded a 278-amino acid protein with a
calculated molecular mass of ~32 kDa. Expression of this protein
in E. coli produced a fully functional recombinant protein
that also catalyzed the enantiospecific conversion of (
)-secoisolariciresinol into (
)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this
overall enzymatic conversion now unambiguously defined, the entire
biochemical pathway to the lignans, secoisolariciresinol and
matairesinol, has been elucidated. Last, both secoisolariciresinol and
matairesinol are metabolized in the gut of mammals, following digestion
of high fiber dietary grains, seeds, and berries, into the so-called
"mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the
onset of breast and prostate cancers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Lignans with various pharmacological and
physiological activities.
Lignans also have important physiological roles in planta, since many function as biocidal agents, feeding deterrents, antioxidants, and allelopathic chemicals (2, 7, 8). Additionally, certain plant species contain lignans with important roles in conferring or defining the quality, color, and durability of various heartwoods; e.g. about 20% of the dry weight of western red cedar (Thuja plicata) heartwood is composed of plicatic acid 9-derived lignans (9, 10).
In species such as Forsythia intermedia, formation of
(+)-pinoresinol 10a, the entry point into its main (if not
exclusive) lignan pathway, occurs by stereoselective coupling of two
molecules of E-coniferyl alcohol 11 (Fig.
2). (+)-Pinoresinol 10a then
undergoes sequential enantiospecific reduction to afford (+)-lariciresinol 12a and ()-secoisolariciresinol
6a, with dehydrogenation of the latter occurring to give
(
)-matairesinol 5a. Depending upon the species involved,
matairesinol 5 is believed to be the precursor of bioactive
molecules such as (
)-podophyllotoxin 1 in
Podophyllum peltatum (11, 12), (
)-trachelogenin
4 in Ipomoea carica (3), and plicatic acid
9 in T. plicata (13) (Fig. 2).
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In this study, the enzymology of formation of ()-matairesinol
5a from (
)-secoisolariciresinol 6a in both
F. intermedia and P. peltatum was investigated.
This resulted in the purification to apparent homogeneity of
(
)-secoisolariciresinol dehydrogenase, the cloning of the
corresponding cDNAs, the expression of functional recombinant
proteins in Escherichia coli, and determination of basic
parameters (Km, Vmax). This
research was conducted as a first step toward obtaining edible
transgenic plants containing elevated levels of matairesinol
5 for health protection, as well as for attaining higher
levels of medicinally active lignans such as podophyllotoxin
1.
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EXPERIMENTAL PROCEDURES |
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Plant Materials-- F. intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St. Paul, MN), and maintained in Washington State University greenhouse facilities or were gifts from the local community. P. peltatum plants, propagated from rhizomes harvested in Virginia, were cultivated in the same greenhouse facilities.
Synthesis of E-[9-3H2]Coniferyl Alcohol 11-- To a solution of coniferyl aldehyde in methanol (1.1 mmol, 5 ml) was added tritiated sodium borohydride (NaB3H4; 13.3 GBq/mmol, 3.7 GBq) at 0 °C. After stirring for 20 min, unlabeled sodium borohydride (NaBH4; 1.3 mmol) was added with the whole stirred for another 20 min. The pH was next adjusted to 6 (by the dropwise addition of 2 N HCl), and the reaction mixture was extracted with diethyl ether (50 ml). The ether solubles were extracted with water, dried (sodium sulfate), and evaporated to dryness in vacuo. The residue was reconstituted in a small amount of ethyl acetate and applied to a short silica gel column (10 × 4-mm inner diameter) eluted with methylene chloride/ethyl acetate (4:1) to afford [9-3H]coniferyl alcohol 11 (1 mmol, 1.21 GBq/mol).
Synthesis of (±)-[9,9'-3H]Secoisolariciresinols 6a/6b-- To [9-3H]coniferyl alcohol 11 (1 mmol in acetone, 7 ml, 1.21 GBq/mol) was added iron (III) chloride hexahydrate (FeCl3·6H2O; aqueous solution, 2.6 mmol, 24 ml) at room temperature. Following stirring for 10 min, the reaction mixture was extracted with diethyl ether (30 ml × 3). The ether solubles were combined, extracted with water (20 ml), dried (sodium sulfate), and evaporated to dryness in vacuo. The residue was reconstituted in a minimum amount of methylene chloride and applied to a silica gel column (15 × 2.5-cm inner diameter) eluted with methylene chloride/diethyl ether (4:1) to give pure (±)-[9,9'-3H]pinoresinols 10a/10b (0.1 mmol, 2.42 GBq/mol, 20% yield). To a stirred solution of (±)-[9,9'-3H]pinoresinols 10a/10b (0.1 mmol, 2.42 GBq/mol, 5 ml) in methanol was added 10% palladium on charcoal (80 mg) under H2. After a 24-h reduction, the catalyst was removed by filtration, washed with methanol (5 ml) with the methanol solubles combined, and evaporated to dryness in vacuo to afford, following preparative thin layer chromatography (ethyl acetate/hexanes/methanol (10:10:1)), (±)-[9,9'-3H]secoisolariciresinols 6a/6b (0.07 mmol, 2.42 GBq/mol, 70% yield).
Synthesis of ()-Lactol 13a--
To (
)-matairesinol
5a (120 mg, 0.34 mmol) in dry tetrahydrofuran (20 ml) was
added dropwise 1 M lithium triethylborohydride (LiEt3BH) in tetrahydrofuran solution (1.1 ml) at 0 °C,
with the resulting suspension stirred for 1 h at this temperature.
To quench the reaction, 2 N HCl was added slowly until the
reaction mixture was of pH ~6. To this was added ethyl acetate (150 ml), with the whole then washed with water (50 ml). The organic
solubles were next dried (sodium sulfate) and evaporated to dryness
in vacuo. The resulting residue was reconstituted in a
minimum amount of ethyl acetate and applied to a silica gel column
(20 × 2-cm inner diameter), eluted with ethyl acetate/hexanes
(1:1 and 2:1) to afford (
)-lactol 13a (72 mg, 60% yield
in two isomers; 3:5 ratio). UV
max (methanol): 229.7, 279.4 nm.; electron impact mass spectroscopy m/z (%): 360 (M+, 12.1), 205 (10.2), 163 (9.2), 137 (100), 122 (8.2);
HRMS m/z: found 360.1560 [M]+, calculated for
C20H24O6: 360.1573; 1H
NMR (CDCl3):
2.36-2.84 (6H, m,
C7,7'H, C8,8'H), 3.53-4.16 (2H, m, C9'H), 3.79, 3.85 (6H, s,
OCH3), 5.25 (1H, C9 H), 6.42-6.84 (6H, m,
Ar-H). 13C NMR (CDCl3):
31.30, 33.81, 38.69, 39.12, 39.58, 43.19, 46.05, 52.17, 53.18, 56.01, 56.18, 72.63, 72.92, 99.15, 103.68, 111.25, 111.29, 111.42, 111.75, 114.30, 114.38, 114.48, 114.55, 121.41, 121.60, 121.77, 131.67, 132.20, 132.52, 132.83, 143.95, 144.11, 144.16, 146.58, 146.69.
Secoisolariciresinol Dehydrogenase Assays--
Assays with
(±)-[9,9'-3H]secoisolariciresinols 6a/6b and
(±)-[Ar-2H]secoisolariciresinols 6a/6b were
carried out as reported elsewhere (12). Assays with ()-lactol
13a as substrate at a final concentration of 55 µM were carried out as described for secoisolariciresinol
6, with matairesinol 5 formation being quantified
using a previously established standard curve.
Chemical Conversion of Enzymatically Formed [9'-3H]Matairesinol 5 into [9'-3H]Secoisolariciresinol 6-- This chemical synthetic procedure was carried out as reported elsewhere (12).
General Procedures for Enzyme Purification--
All
manipulations were carried out at 4 °C with chromatographic eluents
monitored at 280 nm, unless otherwise indicated. Protein concentrations, using -globulin as a standard, were determined by
the method of Bradford (14). Polyacrylamide gel electrophoresis was
performed with Laemmli's buffer system under denaturing or nondenaturing conditions, as well as with gradient gels (4-15%) (15);
proteins were visualized by silver staining (16).
Preparation of Cell-free Extracts-- F. intermedia stems (2 kg) were frozen (liquid N2) and pulverized in a Waring blender (model CB6). The resulting powder was homogenized with Tris-HCl buffer (50 mM, pH 7.5, 2 liters) containing 5 mM dithiothreitol (buffer A). The homogenate was filtered through four layers of cheesecloth into a beaker containing polyvinylpolypyrrolidone (10%, w/v), with the filtrate centrifuged (10,000 × g, 15 min) and the resulting supernatant fractionated with ammonium sulfate. Proteins precipitating between 30 and 60% saturation were recovered by centrifugation (10,000 × g, 30 min) with the pellet then reconstituted in a minimal amount of buffer A.
DEAE Chromatography-- The crude enzyme preparation (445 mg in 90 ml of buffer A; 4 nmol/h/mg protein) was applied to a DEAE-cellulose column (40 × 2.6-cm inner diameter) equilibrated in buffer A. Secoisolariciresinol dehydrogenase was eluted (after washing the column with 25 ml of buffer A) with a linear NaCl gradient (0-2 M in 500 ml) in buffer A at a flow rate of 2.5 ml/min. Active fractions were combined, concentrated by ultrafiltration (Amicon, YM10 membrane) to 50 ml, and dialyzed (25 mM Tris-HCl buffer, pH 7.5, 5 mM dithiothreitol) overnight.
Affinity (2',5'-ADP-Agarose) Chromatography-- The active fractions from the DEAE-cellulose chromatography (201 mg, 14 nmol/h/mg protein) were applied to a 2',5'-ADP-agarose (10 × 1-cm inner diameter) column previously equilibrated in Tris-HCl buffer (25 mM, pH 7.5, 5 mM dithiothreitol). The column was first washed with 20 ml of the same buffer at a flow rate of 1 ml/min and then with 50 ml of buffer A containing 500 mM NaCl, and finally secoisolariciresinol dehydrogenase was eluted with NAD (10 mM) in buffer A. The active fractions were combined and dialyzed 15 h against buffer A.
MonoP (HR 5/20) Column Chromatography-- Active protein (185 µg, 8.4 µmol/h/mg protein) from the preceding step was applied to a MonoP column equilibrated in buffer A, washed with buffer A (8 ml), and eluted with a linear NaCl gradient (0-2 M in 145 ml) in buffer A at a flow rate of 1 ml/min. The active fractions (74 µg, 17.7 µmol/h/mg protein) were combined, dialyzed against buffer A, and then subjected to a second round of MonoP column chromatography using the procedure described above. Secoisolariciresinol dehydrogenase (31 µg, 24.2 µmol/h/mg protein) obtained was next analyzed by SDS-polyacrylamide gel electrophoresis.
cDNA Library Synthesis-- The amplified cDNA libraries individually prepared from young green stems of greenhouse grown F. intermedia plants (var. Lynwood Gold) and from rhizomes of greenhouse grown P. peltatum were constructed as described previously (12, 17). Both were screened for secoisolariciresinol dehydrogenase clones as detailed below.
Secoisolariciresinol Dehydrogenase DNA Probe Synthesis--
The
N-terminal and internal trypsin digest peptide amino acid sequences
were used to construct degenerate oligonucleotide primers (see
"Results and Discussion"). Purified F. intermedia cDNA library DNA (2 ng) (17) was used as template in 100-µl PCRs1 (10 mM
Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 0.2 mM each dNTP, and 2.5 units of Taq DNA polymerase) along with 2.5 pmol each of
primer DEHYF26 and either primer DEHYF30RevA or primer DEHY30RevB (see
"Results and Discussion"). PCR amplification was carried out in a
thermocycler with 35 cycles of 94 °C denaturing for 1 min, 50 °C
annealing for 2 min, and 72 °C extension for 3 min. PCR products
were resolved in 1.5% agarose gels, where a single band of ~200 base
pairs (bp) was obtained. The resulting PCR product was ligated into a
pT7Blue T-vector and transformed into competent NovaBlue cells
according to Novagen's instructions. DNA sequence analysis indicated
that the recombinant plasmid insert coded for the initial internal
trypsin digest fragment IMFSNAGISDPN (see "Results and Discussion")
obtained from the native plant protein. A
BamHI/SpeI fragment of ~200 bp harboring this
insert was excised from the plasmid preparation, purified by running on
a 1% low melting point agarose gel, and used as a probe to screen the
cDNA library. The probe was prepared by boiling the purified DNA
fragment (100 ng) for 10 min, followed by radiolabeling for 20 min at
37 °C using the Amersham Pharmacia Biotech
T7QuickPrime Kit and [-32P]dCTP.
Unincorporated nucleotides were removed from the radiolabeled probe by
passing it through a CENTRI-SPINTM-20 spin column
(Princeton Separations Inc.).
F. intermedia cDNA Library Screening, Excision of Recombinant Phage, and DNA Sequencing-- See Supplemental Material.
Heterologous Expression in E. coli of F. intermedia
Secoisolariciresinol Dehydrogenase--
The E. coli culture
containing the F. intermedia secoisolariciresinol
dehydrogenase clone was grown at 37 °C in 25 ml of Luria-Bertani (LB) medium (18) supplemented with kanamycin (50 µg/ml) to a density
of A600 = 0.5. To this was added isopropyl
thio--D-galactoside to give a final concentration of 0.5 mM (according to the Invitrogen protocol), and the culture
was grown at 18 °C for an additional 20 h. The cells were
pelleted (600 × g, 4 °C, 12 min), resuspended in 5 ml of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM dithiothreitol, and repelleted. The final bacterial
pellet was resuspended in 200 µl of the above buffer and sonicated
4 × 15 s using a Braun-Sonic 2000 sonicator. Next, the
sample was centrifuged for 5 min at 16,000 × g. An
aliquot of the supernatant (2 µl, 10 µg of total protein) was
subjected to SDS-PAGE analysis, whereas another aliquot (50 µl, 250 µg of total protein) was assayed for (
)-secoisolariciresinol dehydrogenase activity (see "Results and Discussion"). (A negative control, prepared from an E. coli culture containing an
unrelated gene encoding phenylcoumaran benzylic ether reductase from
Pinus taeda cloned into the SBET expression vector (19), was
also assayed for dehydrogenase activity). HPLC analysis of the assay mixture revealed the formation of a compound eluting at an elution volume of 17.4 ml in addition to matairesinol 5. This
compound was identified as (
)-lactol 13a. MS
m/z (%) 360 (M+, 14.2), 205 (14.3), 163 (9.4),
137 (100), 122 (10.2). UV
max (methanol): 229.7, 279.4 nm.
P. peltatum Secoisolariciresinol Dehydrogenase Clone Isolation-- See Supplemental Material.
Heterologous Expression of P. peltatum Secoisolariciresinol Dehydrogenase in E. coli-- See Supplemental Material.
Purification of Recombinant P. peltatum Secoisolariciresinol Dehydrogenase-- The crude enzyme (150 mg) in buffer A (5 ml) was subjected to (NH4)2SO4 fractionation with the proteins precipitating between 20 and 60% saturation recovered by centrifugation (15,000 × g; 15 min; 4 °C). The pellet was reconstituted in Tris-HCl buffer (50 mM, pH 7.5) containing 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA (buffer B, 6 ml). The resulting solution, dialyzed 24 h against buffer B, was next applied to a MonoQ HR 10/10 column equilibrated in buffer B. The column was washed with 10 ml of buffer B, and then secoisolariciresinol dehydrogenase was eluted using a linear NaCl gradient (0-400 mM in 130 ml) in buffer B at a flow rate of 1 ml/min. Active fractions were combined, pooled, and dialyzed against buffer B (4 × 500 ml, 30 min each) and further applied to a MonoQ HR 5/5 column equilibrated in buffer B. Secoisolariciresinol dehydrogenase was eluted using the same gradient as described for the MonoQ HR 10/10 column. Active fractions were combined and concentrated to 400 µl, using a Microcon YM 3 microconcentrator (Amicon). The enzyme solution was then applied in 200-µl portions to a Superdex 75 (HR 10/30) column. Gel filtration was performed in buffer B containing 150 mM NaCl at a flow rate of 0.4 ml/min, and the secoisolariciresinol dehydrogenase was eluted with mobile phase (9.4 ml). The collected fractions (0.4 ml) were analyzed by SDS-polyacrylamide gel electrophoresis (25 µl) as well as being assayed for secoisolariciresinol dehydrogenase activity (10 µl). Active fractions were immediately used for pH and temperature optimum determinations and kinetic studies as described below.
pH and Temperature Optima--
To determine the pH optimum of
secoisolariciresinol dehydrogenase, standard assay conditions using
()-lactol 13a as substrate were employed except that the
buffer was replaced with 20 mM MES buffer in the pH range
of 4-6.5, 20 mM Bis-Tris buffer in the pH range of
6.5-9.0, and 20 mM CAPS buffer in the pH range of
9.0-11.5. The temperature optimum was examined in the range between 0 and 80 °C under standard assay conditions at pH 8.8. Assays were
carried out with 10 µg of purified recombinant
secoisolariciresinol dehydrogenase.
Kinetic Parameters--
Initial velocity studies were
individually performed in triplicate experiments, using 20 mM Tris-HCl buffer, pH 8.8, containing the purified
secoisolariciresinol dehydrogenase (10 µg) at six different
()-lactol 13a and (±)-secoisolariciresinol
6a/6b concentrations (between 15 and 167 µM)
with a constant NAD concentration (100 µM). Incubations
were carried out at 20 °C for 1 and 4 min for (
)-lactol
13a and secoisolariciresinol 6, respectively. Kinetic parameters were determined from Lineweaver-Burk plots.
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RESULTS AND DISCUSSION |
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The levels of the health-protecting lignans, matairesinol 5 and secoisolariciresinol 6, are typically very low in foodstuffs commonly used in the western diet. The overall purpose of this study was thus to obtain the gene(s) encoding the enzyme(s) catalyzing the conversion of secoisolariciresinol 6 into matairesinol 5, as a forerunner to designing strategies to obtain transgenic plants with elevated levels of these protective substances in staple western dietary foodstuffs. Indeed, this conversion represents the final step in the biochemical pathway from coniferyl alcohol 11 to matairesinol 5, since all of the preceding steps from coniferyl alcohol 11 to secoisolariciresinol 6 have been fully defined, in terms of the proteins, enzymes, and genes involved (1, 17, 20-23). Moreover, since matairesinol 5 is also a precursor of the antiviral and antitumor lignan, podophyllotoxin 1 (11, 12), the opportunity also availed itself to obtain the corresponding gene(s) encoding secoisolariciresinol dehydrogenase from Podophyllum species; interestingly, Podophyllum sp. are difficult to cultivate, and the native species growing in the wild are being overharvested, particularly in Asia.
In earlier studies using crude cell-free extracts from F. intermedia, the enantiospecific conversion of
()-secoisolariciresinol 6a into (
)-matairesinol
5a had been established, although whether one enzyme or more
was involved was not known (Fig. 2) (24, 25). Accordingly, since
F. intermedia also accumulated several matairesinol
5-derived substances, this plant species was selected
initially as a suitable source of enzyme(s) involved in formation of
the lignan, (
)-matairesinol 5a (26).
Purification and Characterization of ()-Secoisolariciresinol
Dehydrogenase--
In the present study, the F. intermedia
NAD-dependent enzyme responsible for this conversion was
first purified (>6,000-fold) to apparent homogeneity using a
combination of ammonium sulfate precipitation and DEAE-cellulose,
ADP-agarose, and MonoP (HR 5/20) chromatographic steps, respectively
(see Table I). Next, SDS-polyacrylamide gel electrophoretic analysis of the resulting secoisolariciresinol dehydrogenase gave an apparent molecular mass of ~32 kDa (Fig. 3). As for the related
pinoresinol/lariciresinol reductase (17), however, which catalyzes the
two preceding biochemical steps, the corresponding secoisolariciresinol
dehydrogenase was also present in very low abundance (37 µg from 2 kg
of F. intermedia stems).
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The enzyme assays employed for its detection utilized either
(±)-[9,9'-3H]secoisolariciresinols 6a/6b or
[Ar-2H]secoisolariciresinols 6a/6b as
substrates; the (±)-[9,9'-3H]secoisolariciresinols
6a/6b were obtained by iron chloride (FeCl3)-catalyzed coupling of
E-[9-3H]coniferyl alcohol 11 (1.21 GBq/mol) to afford the racemic (±)-[9,9'-3H]pinoresinols
10a/10b, with subsequent reduction (10% palladium on
charcoal, H2) generating the required
(±)-[9,9'-3H]secoisolariciresinols 6a/6b
(2.42 GBq/mg). [Ar-2H]Secoisolariciresinols
6a/6b, on the other hand, were prepared by acid-catalyzed
deuterium exchange of the aromatic ring protons using deuterated
trifluoroacetic acid (CF3CO2D) as previously
described (25); its mass spectrum displayed the expected molecular ion
cluster centered at m/z 364 (Fig.
4A), this being indicative of
two or three hydrogen atoms being replaced by deuterium.
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Verification that the purified secoisolariciresinol dehydrogenase
catalyzed the enantiospecific conversion of ()-secoisolariciresinol 6a into (
)-matairesinol 5a was demonstrated in two different ways: first, via incubation of the enzyme with
(±)-[9,9'-3H]secoisolariciresinols 6a/6b (2.8 µM, 3.4 kBq) in the presence of 40 µM NAD
for 2 h, with unlabeled (±)-matairesinols 5a/5b (3 µg) being added as radiochemical carriers. The resulting radiolabeled
[9'-3H]matairesinol 5 so formed was then
purified by reversed-phase HPLC and subsequently reduced chemically
with lithium aluminum hydride to regenerate secoisolariciresinol
6. This step was necessary, since both (
)- and
(+)-matairesinols 5a and 5b are only partially
resolved by chiral column HPLC. Fig. 5
shows the chiral column HPLC separations of both (
)- and (+)-secoisolariciresinol 6a/6b standards (Fig.
5A) as well as that of the product derived from enzymatic
incubation (Fig. 5B). Thus, since only
(
)-[9'-3H]secoisolariciresinol 6a was
present, this demonstrated that the matairesinol 5 enzymatically produced was only in the (
)-form 5a;
i.e. (+)-secoisolariciresinol 6b had not served
as a substrate. Second, the purified NAD-dependent secoisolariciresinol dehydrogenase converted
[Ar-2H]secoisolariciresinol 6 into
[Ar-2H]matairesinol 5 (Fig. 4B),
i.e. the enzymatic product gave the expected molecular ion
cluster at m/z 360, this being centered 2-3 mass units
higher than that of natural abundance matairesinol 5 (m/z 358) (Fig. 4C) and thus demonstrating an
intact conversion of substrate 6a into 5a.
Together, both results unambiguously established the enantiospecificity and authenticity of the enzymatic conversion catalyzed by
(
)-secoisolariciresinol dehydrogenase.
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Gene Cloning, Functional Expression, and Characterization of
Recombinant ()-Secoisolariciresinol Dehydrogenase from F. intermedia--
Attention was next directed toward obtaining the
encoding gene for (
)-secoisolariciresinol dehydrogenase. Thus,
following the final column chromatographic step (Table I), the F. intermedia 32-kDa protein was subjected to SDS-polyacrylamide gel
electrophoresis and blotted onto a polyvinylidene difluoride membrane,
this then being subjected to Edman degradation analysis (see
Supplemental Material). Additionally, sequences of internal fragments
were obtained by trypsin digestion of the purified secoisolariciresinol dehydrogenase (see Supplemental Material).
Fig. 6 shows the N-terminal and internal
trypsin fragment sequences obtained; the 17-amino acid internal trypsin
digestion sequence (VALITGGASGIGETTAK), which overlapped with the
N-terminal sequence, had strong homology to the N-terminal sequences of
other dehydrogenases when subjected to a BLAST homology search
comparison (27), which included short-chain alcohol dehydrogenases from cowpea (Vigna unguiculata, 82% identity and 93%
similarity) (28), and Nicotiana tabacum (82% identity, 87%
similarity) (29). The other internal trypsin digestion sequence
(LNIMFSNAGISDPNK), however, was also assumed to be in close proximity
to the N-terminal sequence, as projected by comparison of its alignment
with published sequences for alcohol dehydrogenases. Thus, forward
(DEHYF26) and reverse (DEHYF30RevA and -B) degenerate oligonucleotide
primers (Fig. 6) were next synthesized for sequences closest to, and
furthest from, the N terminus, respectively.
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Using the PCR-guided strategy described in the Supplemental Material, with the F. intermedia cDNA library as template (17), a prominent DNA band (~200 bp) was obtained, this being cloned into a pT7 Blue T-vector (Novagen), and transformed into competent NovaBlue (Novagen) E. coli cells. The DNA sequence of this insert established that the internal trypsin digest fragment sequence IMFSNAGISDPN (Fig. 6) was present in the PCR product cloned into the vector.
This 200-bp fragment was next used as a probe in an effort to screen
the amplified F. intermedia cDNA library to obtain the complete cDNA clone. Numerous strong positive hybridization signals were detected, of which 11 were isolated and sequenced. One clone (SDH_Fi321, GenBankTM accession number AF352735) contained a start Met
preceded by a 5'-untranslated region, and its 831-bp open reading
frame, the longest of the sequences obtained, predicted a polypeptide
of 277 amino acids in size and a calculated mass of 32 kDa (see Fig.
7). This is in close agreement with the
estimated band size of the denatured protein as observed by
SDS-polyacrylamide gel electrophoresis analysis of the purified native
plant enzyme (see Fig. 3); the N-terminal sequence of SDH_Fi321 also
matched the N terminus and trypsin fragment amino acid sequences
obtained from the F. intermedia native protein originally
isolated (Fig. 6).
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5' and 3' primers were next designed to introduce NdeI restriction enzyme sites at both ends of the clone to allow for subsequent insertion into the SBET expression vector (30) for overexpression of the protein in E. coli. Deployment of the primers for PCR, with 2 ng of the previously obtained SDH_Fi321 clone plasmid DNA as template, gave a PCR product that was then cloned directly into the TA vector. The dehydrogenase clone was next excised from the construct using the NdeI sites at the 5'- and 3'-ends and cloned into the SBET vector; this construct was then transformed into the E. coli strain BL21(DE3) for heterologous overexpression.
Heterologous Expression of F. intermedia
()-Secoisolariciresinol Dehydrogenase in E. coli--
E.
coli cells harboring the SDH_Fi321/SBET construct were grown until
mid-log phase and then induced with isopropyl
thio-
-D-galactoside, as described under "Experimental
Procedures." The resulting cell-free extract was incubated in
the presence of (±)-[9,9'-3H]secoisolariciresinols
6a/6b (2.8 µM, 3.4 kBq) and 40 µM NAD. After a 2-h incubation, unlabeled
(±)-matairesinols 5a/5b (3 µg) were added as
radiochemical carriers, with the enzyme extract then subjected to
reversed-phase HPLC (Fig. 8). Two
enzymatic products were observed. First, in terms of the enzymatically
generated [9'-3H]matairesinol 5, the
enantiospecificity of its formation was established by subjecting it to
a chemical reduction, using lithium aluminum hydride as before to
afford secoisolariciresinol 6, with the latter being
analyzed by chiral column chromatography (Chiralcel OD, Daicel). It was
thus established that only (
)-secoisolariciresinol 6a was
obtained following chemical reduction, and not the antipode
6b (Fig. 5C); i.e. as expected, only
(
)-matairesinol 5a formation was catalyzed by the
heterologously expressed (
)-secoisolariciresinol dehydrogenase. To
further confirm the authenticity of this conversion, assays were also
performed for 2 h with
(±)- [Ar-2H]secoisolariciresinols 6a/6b
(molecular ion cluster centered at m/z 364) as substrates
(0.55 µmol) with 40 µM NAD as cofactor, in the presence
of the cell-free extract. The enzymatically formed [Ar-2H]matairesinol 5 was then purified and
submitted to mass spectrometric analysis. This gave the expected
molecular ion cluster centered at m/z 362 (data not shown)
as noted previously for the native protein (see Fig. 4B).
Thus, both the F. intermedia native protein and the
heterologously expressed (
)-secoisolariciresinol dehydrogenase
catalyzed the same enantiospecific reaction. Note also that with a
corresponding negative control (expression of an unrelated gene,
phenylcoumaran benzylic ether reductase, cloned into the SBET vector
(19)), no enzymatic activity of any type was detected using
(±)-secoisolariciresinols 6a/6b as substrates (data not
shown).
|
In addition to the expected enzymatic product ()-matairesinol
5a, a second product was also noted during HPLC analysis with an elution volume of 17.5 ml. Attention was next directed toward
the identification of this unknown compound. This was found to have a
molecular ion at m/z 360, with a base peak at m/z
137 and an UV absorption spectrum with maxima at
229.7 and 279.4 nm, respectively. Together, these spectroscopic data as well as its
chromatographic behavior suggested that the compound might be the
corresponding lactol 13. That this was indeed the case was
established by the chemical synthesis of (
)-lactol 13a, obtained via reduction of (
)-matairesinol 5a with lithium triethylborohydride (LiEt3BH); i.e. the
resulting synthetic product displayed identical HPLC chromatographic
behavior, as well as UV and mass spectra, to that of the enzymatic
product. Furthermore, synthetic 13 was additionally
characterized by 1H and 13C NMR spectroscopic
analyses, as well as by HRMS (see "Experimental Procedures"). Thus,
its identification unequivocally established that the
enantiospecific conversion of (
)-secoisolariciresinol 6a
to (
)-matairesinol 5a proceeded via the intermediary (
)-lactol 13a (see Fig.
9).
|
Cloning, Expression, and Characterization of
()-Secoisolariciresinol Dehydrogenase Homologue from P. peltatum--
As indicated earlier, both P. peltatum and
Podophyllum hexandrum are sources of the important antiviral
and antitumor lignan podophyllotoxin 1, and a recent study
using radiolabeled substrates also demonstrated matairesinol
5 to be a precursor of podophyllotoxin 1 (12);
i.e. the biochemical pathway to matairesinol 5 in
Podophyllum sp. is the same as that for
Forsythia. Accordingly, the next objective was to obtain the gene encoding the (
)-secoisolariciresinol dehydrogenase from a
P. peltatum cDNA library. Thus, PCR screening, using the
degenerate primers (DEHYF26 forward and DEHYF30Reva reverse), was again
employed, and this gave a 224-bp PCR band 70% similar and 50%
identical to the homologous region of the F. intermedia
secoisolariciresinol dehydrogenase gene. This was used to design the
internal gene specific forward and reverse primers PPD7FOR and PPD7REV
(see Fig. 10), which were then coupled
with T7 or T3 primers, respectively, in a subsequent PCR using the
P. peltatum rhizome cDNA library as template. The PCR
products obtained thus provided the complete 5' and 3' segments of the
gene, and these were next used to design the gene-specific 5' forward
and 3' reverse primers PPDNTER and PPDCTER (see Fig. 10). The resulting
PCR product yielded a clone encoding the entire P. peltatum
secoisolariciresinol dehydrogenase sequence SDH_Pp7 (GenBankTM
accession number AF352734; (Fig. 10), and this 834-bp gene encoded a
278-amino acid protein having 60% similarity and 51% identity to that
of the F. intermedia secoisolariciresinol dehydrogenase. The
P. peltatum gene was next cloned into an Invitrogen pTRCHIS2
TOPO TA vector and transformed into TOP10 E. coli cells. (This system was used to obtain a more rapid cloning and expression of
this gene compared with the previous SBET vector system used for the
F. intermedia secoisolariciresinol dehydrogenase clone). Induction of a culture of transformed cells with isopropyl
thio-
-D-galactoside (described in the Supplemental
Material) produced a 32-kDa soluble recombinant protein, capable of
stereospecifically converting (
)-[9,9'-3H]secoisolariciresinol 6a into
both (
)-[9'-3H]lactol 13a and
(
)-[9'-3H]matairesinol 5a in the presence of
NAD; the corresponding (+)-enantiomers did not serve as substrates
(data not shown).
|
The recombinant P. peltatum secoisolariciresinol
dehydrogenase was next purified to apparent homogeneity using a
combination of anionic and gel filtration chromatographic steps, and
this preparation was used for determination of the pH and temperature optima, as well as kinetic parameters (Km and
Vmax values). ()-Lactol 13a was
initially used as substrate, since only a single dehydrogenative step
was involved.
The pH optimum was examined over the pH range 4-11.5 and reached a
maximum at about pH 8.8. At this optimal pH, the apparent temperature
optimum was established to be ~20 °C. Additionally, kinetic
properties using racemic ()-lactol 13a as a substrate were
examined, with initial velocity studies using substrate concentrations ranging between 15 and 167 µM, while keeping the NAD
concentration constant (100 µM). Apparent
Km values ~160.2 ± 0.8 µM with apparent maximum
velocities ~7.1 ± 0.02 × 103 (expressed as
mmol/min/mg protein) were obtained from Lineweaver-Burk plots;
i.e. typical Michaelis-Menten kinetics were observed.
Next, racemic (±)-secoisolariciresinols 6a/6b were used as
substrates over the same range of concentrations as for the lactol
13. Given the potential for complexity due to the
bifunctional nature of the enzyme (diol lactol
lactone), kinetic parameters were studied as a function of secoisolariciresinol 6 depletion. At high substrate concentrations (
75
µM), the reaction followed typical Michaelis-Menten
kinetics. However, at lower substrate concentrations, there was a
deviation from classical Michaelis-Menten kinetics, the basis of which
will be investigated in the future. Of particular note, the overall
conversion was stereospecific, using only the (
)-enantiomer, and the
build-up of lactol 13 at high substrate concentrations (
50
µM) strongly suggested that the second dehydrogenative
step is rate-limiting.
Sequence Homology Comparisons--
Comparisons using the GAP
program (31) revealed significant homology of the F. intermedia and P. peltatum secoisolariciresinol dehydrogenase genes to that of a drought-induced probable short-chain alcohol dehydrogenase of unknown function from cowpea (28)
(i.e. 63 and 60% similarity and 55 and 49% identity,
respectively). Interestingly, the F. intermedia SDH_Fi321
and P. peltatum SDH_Pp7 clones had an N-terminal initiation
residue at a location similar to that of cowpea (CPRD12; Fig.
11), although differing by one amino
acid. This association helped us to approximate the locations of the
amino acid fragments that we had obtained from the native protein,
which was instrumental in the approach to design degenerate primers.
Moreover, the NAD-binding site is highly conserved for all of the
proteins compared in Fig. 11 in accordance with previous observations
with other NAD-dependent dehydrogenases. It was suggested that the preservation of both the structure and function of the NAD-binding domains, as is illustrated by the examples compared in Fig.
11, indicates an evolutionary relationship based on an ancestral
NAD-binding protein (32). Indeed, there are now more than 100 different
members of this NAD-dependent dehydrogenase family known
(32), with enzymes of this type being commonly found in both plant and
animal kingdoms. Interestingly, neither of the two secoisolariciresinol
dehydrogenase sequences contain any secretory pathway signal sequences,
in accordance with their presumed cytosolic character.
|
Conclusion--
The isolation and characterization of the two
secoisolariciresinol dehydrogenase genes from F. intermedia
and P. peltatum, involved in the formation of
()-matairesinol 5a, was of considerable interest for two
reasons: the first is that this represents the final step in the
biochemical pathway to the phytoestrogenic, health-protecting, lignan,
matairesinol 5, and thus all steps from coniferyl alcohol
11 are fully characterized. The second reason is that we are
now poised to manipulate the levels of these important compounds
whether for disease prevention (i.e. matairesinol
5 and secoisolariciresinol 6) or for curative
purposes (e.g. for enhanced podophyllotoxin 1 formation); i.e. future work will be directed to metabolic
engineering of the levels of secoisolariciresinol 6 and
matairesinol 5 in plant foodstuffs such as vegetables,
grains, and fruits or as supplements for processed food items. As
stated earlier, matairesinol 5 and secoisolariciresinol
6 are typically present at very low levels in these
economically important crops; thus, engineering their biosynthetic
pathways would provide a facile source of these beneficial lignans in
staple dietary foodstuffs. Related studies will be directed also toward
manipulating the levels of podophyllotoxin 1 in
Podophyllum species or introducing the corresponding genes
into organisms that can be more readily cultivated.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Gerhard Munske for assistance with amino acid sequencing.
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FOOTNOTES |
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* This research was supported in part by United States Department of Agriculture Grant 99-35103-8037, the United States Department of Agriculture McIntire-Stennis Program, and the Lewis B. and Dorothy Cullman and G. Thomas Hargrove Center for Land Plant Adaptation Studies as well as a United States Department of Energy-National Science Foundation Plant Biotechnology Research and Training Center graduate assistantship (to H. C. P.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF352734 and AF352735.
The on-line version of this article (available at
http://www.jbc.org) contains additional experimental materials and references.
To whom correspondence should be addressed. Tel.: 509-335-2682;
Fax: 509-335-7643; E-mail: lewisn@wsu.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M008622200
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; bp, base pair(s); MES, 2-(N-morpholino)ethanesulfonic acid); Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-2(hydroxymethyl)-1,3-propanediol; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid.
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