Conversion of (3 S,4 R)-tetrahydrodaidzein to (3 S)-equol by THD reductase: Proposed mechanism involving a radical intermediate

Article abstract of DOI:10.1021/bi100465y

To elucidate the mechanism of (3S)-equol biosynthesis, (2,3,4-d ₃)-trans-THD was synthesized and converted to (3S)-equol by THD reductase in Eggerthella strain Julong 732. The position of the deuterium atoms in (3S)-equol was determined by ¹H NMR and ²H NMR spectroscopy, and the product was identified as (2,3,4_α-d ₃)-(3S)-equol. All the deuterium atoms were retained, while the OH group at C-4 was replaced by a hydrogen atom with retention of configuration. To explain the deuterium retention in this stereospecific reduction, we propose a mechanism involving radical intermediates.

Full text of DOI:10.1021/bi100465y

5582 Biochemistry 2010, 49, 5582–5587
DOI: 10.1021/bi100465y
Conversion of (3S,4R)-Tetrahydrodaidzein to (3S)-Equol by THD Reductase:
Proposed Mechanism Involving a Radical Intermediate†
Mihyang Kim,‡ E. Neil G. Marsh,§ Soo-Un Kim,*, and Jaehong Han*,‡
‡Metalloenzyme Research Group and Department of Biotechnology, Chung-Ang University, Anseong 456-756, Korea,
§Department of Chemistry and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, and
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences,
Seoul National University, Seoul 151-921, Korea
Received March 29, 2010; Revised Manuscript Received May 4, 2010
ABSTRACT: To elucidate the mechanism of (3S)-equol biosynthesis, (2,3,4-d3)-trans-THD was synthesized and
converted to (3S)-equol by THD reductase in Eggerthella strain Julong 732. The position of the deuterium
atoms in (3S)-equol was determined by 1H NMR and 2H NMR spectroscopy, and the product was identified
as (2,3,4R-d3)-(3S)-equol. All the deuterium atoms were retained, while the OH group at C-4 was replaced by a
hydrogen atom with retention of configuration. To explain the deuterium retention in this stereospecific
reduction, we propose a mechanism involving radical intermediates.
Isoflavones, predominantly found in the leguminous plants,
are healthy natural dietary phytoestrogens (1-3). Daidzein (40,7-
dihydroxyisoflavone) is one of the major isoflavones in soybean,
which is present mostly in the glucoside form, daidzin. This com-
pound has recently attracted a great deal of attention because of
its various beneficial effects for human health, including estro-
genic (4), anticancer (5), antioxidant (6, 7), and cardioprotective
activities (8). In the intestine, the glycosidic linkage of daidzin is
cleaved by endogenous microorganisms, and some of the result-
ing daidzeins are absorbed into the bloodstream. The remaining
daidzeins are further metabolized to DHD1 (40,7-dihydroxyiso-
flavanone), THD (40,7-dihydroxyisoflavan-4-ol), and finally
(3S)-equol [(3S)-40,7-dihydroxyisoflavan], which has the most
potent estrogenic effect among the daidzein-derived metabolites
(Figure 1) (9).
Although several human intestinal bacteria that can metabo-
lize daidzein or DHD have been described (10-15), the chemistry
of the bioconversion remains poorly understood, mainly because
of the extreme oxygen sensitivity of the microbes and the enzymes.
Recently, the metabolic pathway and stereochemistry involved in
the transformation of DHD to (3S)-equol by the anaerobic
intestinal bacterium, Eggerthella strain Julong 732, were deter-
mined (16). Reduction of DHD produces only one of the THD
stereoisomers, indisputably assigned as (3S,4R)-THD (17), which
is then converted to (3S)-equol by THD reductase. Apparently,
the reduction of the C-4 hydroxyl group required to form
(3S)-equol from (3S,4R)-THD is catalyzed by a single enzyme,
which requires NADPH and the other unidentified cofactor for
the electron transfer (18).
The originally proposed biosynthetic pathway for the (3S)-
equol biosynthesis postulated that DE (40,7-dihydroxyisoflav-
3-ene) was formed as an intermediate, probably through the
cis-elimination reaction (Figure 2a) (19). However, DE could not
be metabolized into (3S)-equol by Julong 732 (16), and in fact,
DE was identified only from the urine sample (20). Recently,
Kim et al. suggested a mechanism involving the formation of a
carbocation intermediate to explain the unprecedented reductive
isomerization reaction (16). In this mechanism, the Lewis acid-
assisted secondary carbocation formation was proposed to
facilitate the 1,2-hydride shift to form a more stable tertiary
carbocation at C-3 (21). The carbocation mechanism could be
still consistent with the newly determined absolute configura-
tion of the substrate THD, if the subsequent hydride addition is
stereospecific (Figure 2b). Alternatively, simple nucleophilic
substitution of the hydroxyl group with hydride at the C-4 center
would produce (3S)-equol (Figure 2c). However, in this instance,
the assistance by a Brønsted acid is required because hydroxide is
generally a poor leaving group (22). While two suggested mecha-
nisms (Figure 2b,c) are possible with varying degrees of feasi-
bility, the added hydride would end up at a different prochiral
position in the (3S)-equol metabolites. Therefore, biotransforma-
tion of (2,3,4-d3)-trans-THD by THD reductase in Eggerthella
strain Julong 732 was performed to check the validity of the
proposed mechanisms.2
†This work was supported by Korean Research Foundation Grant
KRF-2008-331-F00014 funded by the Korean Government (MOEHRD).
*To whom correspondence should be addressed. J.H.: telephone,
þ82-31-670-4830; fax, þ82-31-675-1381; e-mail, jaehongh@cau.ac.kr.
S.-U.K.: telephone, þ82-2-880-4642; fax, þ82-2-873-3112; e-mail,
soounkim@snu.ac.kr.
EXPERIMENTAL PROCEDURES
Daidzein was purchased from Indofine Co. (Somerville, NJ).
GAM was from Nissui Pharmaceutical Co. (Tokyo, Japan).
Ammonium formate-d5 and methol-d4 were from CDN isotopes
(Pointe-Claire, QC). Pd/C (10% Pd), DMSO, DMSO-d6, and
1Abbreviations: DE, dehydroequol; DHD, dihydrodaidzein; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; GAM, Gifu
anaerobic medium; EI-MS, electron impact ionization mass spectro-
metry; HPLC, high-performance liquid chromatography; KCCM,
Korean Culture Center of Microorganisms; NADPH, nicotinamide
adenine dinucleotide phosphate; NMR, nuclear magnetic resonance;
SAM, S-adenosylmethionine; THD, tetrahydrodaidzein; UV, ultra-
violet; VCD, vibrational circular dichroism.
2Extreme oxygen sensitivity and catalytic activity loss during the
purification hindered the isolation of the enzyme.
r
pubs.acs.org/Biochemistry
Published on Web 06/01/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 26, 2010 5583
DMF were purchased from Aldrich (St. Louis, MO). HPLC-
grade solvents of acetonitrile, ethyl acetate, methanol, and acetic
acid were obtained from Fisher (Pittsburgh, PA).
EI-MS spectra of the compounds were recorded with a JMS-
AX505WA instrument (JEOL, Tokyo, Japan) at 70 eV with an
ion source temperature of 280 °C in positive ion mode. 1H and
13C NMR spectra of the compounds in DMSO-d6 were recorded
at 400 and 100 MHz, respectively, on a JNM-LA400 instrument
(JEOL) at 296 K. 2H NMR spectra were recorded in DMSO at
60 MHz on the same instrument.
Chemical Synthesis and Purification of (2,3,4-d3)-trans-
Tetrahydrodaidzein and (2,3,4,4-d4)-Equol. (2,3,4-d3)-trans-
THD and (2,3,4,4-d4)-equol were synthesized from daidzein by
catalytic hydrogenation according to the published method with
a slight modification (23). The reduction of daidzein was con-
ducted in the presence of ammonium formate-d5 and Pd/C in an
inert atmosphere glovebox under nitrogen. Daidzein (260 mg,
0.90 mmol), Pd/C (255 mg), and NH4HCO3-d5 (252 mg,
200 mmol) were dissolved in MeOH-d4 (20 mL). The reaction
mixture was stirred at 65 °C for 1 h, and the reaction was
monitored by HPLC. For monitoring, a Prostar HPLC system
(Varian, Walnut Creek, CA) equipped with a photodiode array
detector (Prostar 330, Varian) operating at 280 nm and a C18
reversed-phase column (Spherisorb 5 μm ODS2, 4.6 mm ꢀ
250 mm, Clwyd, U.K.) were employed. The mobile phase was
composed of 10% acetonitrile in 0.1% acetic acid (A) and 90%
acetonitrile in 0.1% acetic acid (B). The elution profile started
with an A:B ratio of 80:20 (v/v) for 3 min and then linearly
changed to 20:80 (v/v) over 12 min. The flow rate was 1 mL/min.
The reaction mixture was then dried on a rotary vacuum
evaporator after filtration and dissolved in DMF for HPLC
purification on the semipreparative C18 reversed-phase column
(Spherisorb 5 μm ODS2, 10 mm ꢀ 250 mm). The mobile phase
was composed of 100% water (A) and 100% acetonitrile (B), and
the elution profile was the same as that determined via analytical
HPLC. The flow rate was 3 mL/min for preprative HPLC.
(2,3,4-d3)-trans-THD: 1H NMR [(CD3)2SO, 400 MHz] δ 4.08
(s, 0.5H, H-2R), 4.13 (s, 0.5H, H-2β), 5.17 (br s, 1H, OH), 6.16
(d, J = 2.4 Hz, 1H, H-8), 6.35 (dd, J = 10.8, 2.4 Hz, 1H, H-6),
6.68 (d, J = 8.4 Hz, 2H, H-30), 7.04 (d, J = 8.4 Hz, 2H, H-20),
7.17 (d, J = 8.4 Hz, 1H, H-5); 13C NMR [(CD3)2SO, 100 MHz]
δ 45.50 (C-3), 67.83 (C-2), 68.35 (C-4), 102.28 (C-8), 108.66 (C-6),
115.52 (C-30), 118.04 (C-4a), 129.40 (C-20), 130.36 (C-5), 130.75
(C-10), 155.36 (C-8a), 156.47 (C-40), 157.93 (C-7).
(2,3,4,4-d4)-Equol: 1H NMR [(CD3)2SO, 400 MHz] δ 3.86
(s, 0.5H, H-2β), 4.10 (s, 0.5H, H-2R), 6.16 (d, J = 2.4 Hz, 1H,
H-8), 6.27 (dd, J = 10.8, 2.4 Hz, 1H, H-6), 6.71 (d, J = 8.4 Hz,
2H, H-30), 6.84 (d, J = 8.4 Hz, 1H, H-5), 7.09 (d, J = 8.4 Hz, 2H,
H-20), 9.15 (br s, 1H, OH), 9.27 (br s, 1H, OH).
(3S)-Equol: 1H NMR [(CD3)2SO, 400 MHz] δ 2.75 (ddd,
F
IGURE 1: Metabolic pathway leading to the production of (3S)-
equol fromdaidzein. The DHD to(3S)-equolpathway ismetabolized
by unidentified enzymes in Eggetherlla sp. Julong 732. The stereo-
chemistry of DHD relevant to (3S,4R)-THD production is currently
not known.
JH2R,4R = 2.3 Hz, JH3,4R = 5.5 Hz, JH4R,4β = 15.6 Hz, 1H, H-4R),
2.81 (dd, JH3,4β = 10.5 Hz, JH4R,4β = 15.6 Hz, 1H, H-4β), 2.97
(dddd, JH2R,3 = 3.7 Hz, JH2β,3 = 10.5 Hz, JH3,4β = 10.5 Hz,
F
IGURE 2: Possible mechanisms for the conversion of (3S,4R)-THD to (3S)-equol. (a) Base-catalyzed formation of DE and subsequent hydride
addition at C-4. (b) Lewis acid-catalyzed secondary carbocation formation followed by a 1,2-hydride shift and hydride addition at C-3. (c) Direct
hydride addition at C-4 by nucleophilic substitution.

5584 Biochemistry, Vol. 49, No. 26, 2010
Kim et al.
F
IGURE 3: EI-MS spectra of (a) (2,3,4β-d3)-(3S)-equol and (b) equol. The insets show fragmentation patterns.
J
H3,4R = 5.5 Hz, 1H, H-3), 3.88 (dd, JH2R,2β = 10.5 Hz, JH2β,3
10.5 Hz, 1H, H-2β), 4.12 (ddd, JH2R,2β = 10.5 Hz, JH2R,3 = 3.7 Hz,
H2R,4R = 2.3 Hz, 1H, H-2R), 6.16 (d, J = 2.4 Hz, 1H, H-7), 6.27
=
and filtered, followed by evaporation to dryness on a rotary
vacuum evaporator. The metabolites were then dissolved in DMF
for HPLC purification on the semipreparative C18 reversed-phase
column under the same condition described in the above section.
The metabolite of (2,3,4-d3)-trans-THD was dissolved in MeOH
for EI-MS analysis and dissolved in DMSO-d6 and DMSO for
1H and 2H NMR spectroscopy, respectively.
J
(dd, J = 10.8, 2.4 Hz, 1H, H-6), 6.71 (d, J = 8.4 Hz, 2H, H-30),
6.84 (d, J = 8.4 Hz, H, H-5), 7.09 (d, J = 8.4 Hz, 2H, H-20), 9.13
(s, 1H, OH), 9.25 (s, 1H, OH).
Bacterial Culture and Biotransformation. The culturing of
Julong 732 and biotransformations were conducted in a Concept
400 anaerobic chamber (Ruskin Technology, Leeds, U.K.) under
an atmosphere of 5% CO2, 10% H2, and 85% N2. The stock of
Eggerthella sp. Julong 732 (KCCM 10490), preserved in liquid
nitrogen, was thawed and incubated on a GAM agar plate (15%
agar) for 4 days. A single colony was then transferred to the
GAM broth. Two milliliters of the seed culture was added to 200 mL
of GAM broth and incubated for 1 day, after which 40 mL of the
culture was inoculated into 800 mL of GAM broth. When the
optical density at 600 nm reached 0.05, the synthesized (2,3,4-d3)-
trans-THD substrate at 10 mM in DMF was added to the 800 mL
culture to achieve a final concentration of 0.1 mM. After being
incubated for 48 h, the culture was extracted with ethyl acetate
RESULTS
With the newly established DHD f (3S,4R)-THD f (3S)-
equol pathway (16), the mechanism of THD reduction was
investigated by means of a deuterium-labeled substrate. Chemical
synthesis of (2,3,4-d3)-trans-THD and (2,3,4,4-d4)-equol was
successfully achieved through the catalytic hydrogenation of
daidzein, and the products were isolated by HPLC. The HPLC
retention time and UV spectra were identical to those of the
unlabeled compounds. trans-THD was produced as a racemic
mixture of (3R,4S)- and (3S,4R)-THD. Further stereochemical
resolution of the racemic trans-THD was not pursued, because
only the (3S,4R) isomer is the substrate of THD reductase (16).

Article
Biochemistry, Vol. 49, No. 26, 2010 5585
F
IGURE 5: 2H NMR spectra of (a) (2,3,4,4-d4)-equol and (b) (2,3,4R-d3)-
(3S)-equol.
F
IGURE 4: Comparisonof 1H NMR spectra of(2,3,4R-d3)-(3S)-equol
and (3S)-equol.
DE, which was not observed. Second, reduction of the C-4
hydroxyl group proceeded with retention of configuration, which is
inconsistent with an SN2-type concerted hydride reduction mechan-
ism that would produce (2,3,4β-d3)-(3S)-equol (Figure 2c). Further-
more, we could not observe any of the possible reaction inter-
mediates that might arise from a multistep transformation, such as
(3R)-equol, DE, or (3S,4S)-THD. Therefore, whereas the reduction
of C-4 could involve either a cationic, anionic, or radical mechan-
ism, the results of the deuterium labeling experiment make the
recently described carbocation mechanism unlikely. A mechanism
involving the initial formation of a benzylic carbanion intermediate
also seems unlikely because abstraction of a proton at C-4 would
not be energetically favorable.
However, a radical mechanism for the conversion of (3S,4R)-
THD to (3S)-equol has a mechanistic precedent in the reactions
catalyzed by ribonucleotide reductases, which are all radical
enzymes (26, 27). In particular, these enzymes all cleave the
30-C-H bond of the ribose to facilitate reduction of the 20-OH
group, which occurs with retention of stereochemistry. There-
fore, we propose a similar mechanism for THD reductase that
accounts for the stereochemical course of the reaction.
Comparison of the 1H NMR spectra of the deuterated and
unlabeled THDs allowed the identification the deuterium label
positions (24, 25). The 1H NMR spectrum of (2,3,4-d3)-trans-THD
is characterized by singlet peaks for H-2R and H-2β at δ 4.10 and
4.17, respectively, due to the nonstereospecific incorporation of a
single deuterium at C-2 (that occurs during the reduction of the
planar daidzein precursor) and the lack of signals for H-3 and H-4.
Deuterium-labeled (2,3,4-d3)-trans-THD was reacted with
Eggerthella sp. Julong 732 (KCCM 10490) under anaerobic
conditions. The isolated (3S)-equol had the same retention time
on HPLC and exhibited an UV-vis spectrum identical to that of
the authentic standard. When the metabolite was subjected to
mass spectrometry, the molecular ion peak at m/z 245 corres-
ponding to the molecular ion of d3-(3S)-equol was identified. The
fragmentation pattern of the metabolite was also similar to that
of (3S)-equol (Figure 3), implying the deuterium was positioned
at C-3, C-4, and possibly C-2. The precise positions of the
deuterium labels on d3-(3S)-equol were determined by NMR
1
spectroscopy (Figure 4). In the H NMR spectrum, the proton
In this mechanism, a protein-based radical (Figure 6), X•
(by analogy with ribonucleotide reductase this would be a thiyl
radical), initially abstracts hydrogen from the C-3 position to give
a relatively stable benzylic radical (A). The radical at C-3
facilitates the loss of the C-4 hydroxyl group to form a delocalized
radical cation (B). Subsequent reduction of this species by a
hydride donor can then occur at C-4 with retention of stereo-
chemistry as required by the deuterium labeling pattern (C).
Lastly, addition of the C-3 hydrogen to the same re-face of the
molecule would complete the catalytic cycle to generate (3S)-
equol. Interestingly, when the substrate analogue of (3S,4R)-
isoflavan-4-ol, (3S,4R)-THD without the 40,7-dihydroxyl groups,
was reacted with the THD reductase under the same reaction
conditions, the expected reduction product of (3S)-isoflavan was
not isolated. Therefore, it appears the two OH groups are
important for the resonance stabilization of the reaction inter-
mediate.
signals for H-4R at δ 2.81 and H-3 at δ 2.97 were missing, and
the signals for the two H-2 protons at δ 3.88 and 4.12 were
reduced in intensity by half. Therefore, the product was assigned
2
as (2,3,4R-d3)-(3S)-equol. Furthermore, the H NMR spectrum
of the labeled (3S)-equol (Figure 5) clearly showed the peaks
missing from the 1H NMR spectrum, finally confirming the
labeling positions.
DISCUSSION
The conversion of (2,3,4-d3)-trans-(3S,4R)-THD to (2,3,4R-d3)-
(3S)-equol has led us to the following conclusions. First, no
deuterium labels on the C-ring of (3S,4R)-THD were lost during
the reduction by THD reductase. This strongly suggests a
mechanistically concerted or tightly coupled reduction of the C-4
center. Accordingly, the carbocation mechanism described in
Figure 2b appears to be invalid because otherwise the (2,4,4-d3)-
(3S)-equol product would have been isolated. Here, we have to
point out that if the secondary carbocation in Figure 2b is stable
enough to prevent a 1,2-hydride shift due to the resonance
stabilization of benzylic cation at C-4, then the retention of the
label at C-3 would be reasonable. However, the benzylic carbo-
cation might also be expected to undergo β-elimination to form
In conclusion, deuterium labeling studies have uncovered the
unusual stereospecific course of the (3S)-equol biosynthesis
catalyzed by THD reductase, which is inconsistent with the
previous mechanistic proposals for this enzyme. We suggest a
chemically reasonable mechanism, analogous to that of ribo-
nucleotide reductase, that involves a radical cationic intermediate

5586 Biochemistry, Vol. 49, No. 26, 2010
Kim et al.
FIGURE 6: Suggested mechanism for formation of (3S)-equol from (3S,4R)-THD by the THD reductase. The depicted mechanism entails radical
intermediates and a radical initiating cofactor, but it does not necessarily follow the SAM-utilizing ribonucleotide reductase mechanism.
to explain this unprecedented biochemical reaction. We note that
many unusual microbial transformations of the natural pro-
ducts have been recently found to involve radical enzymes, often
involving SAM (S-adenosylmethionine), and it seems highly
plausible that THD reductase, the genes for which have yet to
be cloned and sequenced, could be a member of this growing
class of enzymes (28). Furthermore, THD reductase activity is
extremely oxygen-sensitive, which is another hallmark of all
radical SAM enzymes. Currently, the isolation and characteriza-
tion of the THD reductase are being studied.
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SUPPORTING INFORMATION AVAILABLE
HPLC chromatogram, NMR spectra of trans-THD and
equol, and EI-MS spectrum of (2,3,4,4-d4)-equol. This material
is available free of charge via the Internet at http://pubs.acs.org.
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of a novel equol-producing bacterium from human feces. Biosci.,
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18. Song, D.-G. (2007) Studies on S-equol producing enzyme from
anaerobic bacterium Julong 732. Master’s Thesis, Seoul National
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