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Applied and Environmental Microbiology, May 2009, p. 3062-3068, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02058-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Stereospecific Biotransformation of Dihydrodaidzein into (3S)-Equol by the Human Intestinal Bacterium Eggerthella Strain Julong 732

Mihyang Kim,¹ Su-Il Kim,¹ Jaehong Han,² Xiu-Ling Wang,³ Dae-Geun Song,⁴ and Soo-Un Kim^1,5*

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea,¹ Metalloenzyme Research Group and Department of Biotechnology, Chung-Ang University, Anseong 456-756, South Korea,² College of Life Science, Agricultural University of Hebei, Baoding 071001, China,³ Natural Products Research Center, Korea Institute of Science and Technology Gangneung Institute, Gangneung 210-340, South Korea,⁴ Plant Metabolism Research Center, Kyung Hee University, Yongin 446-701, South Korea⁵

Received 5 September 2008/ Accepted 10 March 2009

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Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3S)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium Eggerthella strain Julong 732 was studied. THD was synthesized by catalytic hydrogenation, and each stereoisomer was separated by chiral high-performance liquid chromatography. Circular dichroism spectroscopy was used to elucidate the absolute configurations of four synthetic THD stereoisomers. Rapid racemization of DHD catalyzed by Julong 732 prevented the substrate stereospecificity in the conversion of DHD into THD from being confirmed. The absolute configuration of THD, prepared by reduction of DHD in the cell-free incubation, was assigned as (3R,4S) via comparison of the retention time to that of the authentic THD by chiral chromatography. Dehydroequol (DE) was unable to produce the (3S)-equol both in the cell-free reaction and in the bacterial transformation, negating the possible intermediacy of DE. Finally, the intermediate (3R,4S)-THD was reduced into (3S)-equol by the whole cell, indicating the inversion of stereochemistry at C-3 during the reduction. A possible mechanism accounting for the racemization of DHD and the inversion of configuration of THD during reduction into (3S)-equol is proposed.

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Isoflavones are natural dietary phytoestrogens mainly occurring in the leguminous plants, such as soybean. Daidzein and genistein, two major isoflavones in soybean, have received a considerable attention due to their bioactivities beneficial to the human health, including estrogenic (9), anticancer (14), antioxidant (1, 21), and cardioprotective (11) activities. Recently, special interest has been focused on the biological effects of the daidzein metabolites, which are being actively studied for drug development (5, 16).

Daidzein is known to be metabolized in the human intestine by the resident microflora, and various metabolites, such as dihydrodaidzein (DHD), 7,4'-dihydroxyisoflavan-4-ol (tetrahydrodaidzein; THD), 7,4'-dihydroxyisoflav-3-ene (dehydroequol; DE), O-desmethylangolensin (O-DMA), and equol, are detected in the human urine (Fig. 1) (6, 7, 10). Among the metabolites, (3S)-equol has about 100 times higher estrogenic activity than daidzein itself (15). However, only about 30 to 50% of humans can produce equol from daidzein (12). In addition, a high correlation was found between the beneficial effects on females by soy food intake and the presence of equol in their urine (4). Therefore, the ability to metabolize daidzein into equol conferred by the intestinal microflora in human is regarded as a hallmark of daidzein responsiveness (3, 34).

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FIG. 1. Proposed pathway for isoflavone daidzein reduction by intestinal microflora leading to equol formation. The absolute configuration of THD is depicted as (3R,4S) according to the conclusion of the present study.

The daidzein metabolic sequence has been proposed based on the presence of various metabolites of daidzein produced by the human intestinal bacteria; daidzein is reduced into DHD, then into THD and DE, and finally into (3S)-equol in sequential reactions (Fig. 1) (7, 10). However, the pathway and the individual reactions in the pathway have not been fully elucidated partly due to the unavailability of pure microbial isolates.

To confirm the proposed metabolic pathway of the human intestinal microflora, attempts have been made to isolate the daidzein-metabolizing bacterial phenotype from human feces. The reduction of daidzein into equol through the cooperation of the microfloral community in the human intestine is thought to be likely and was demonstrated by using the whole microflora from human (2) and monkey (23) feces. However, daidzein metabolism by the whole-rat intestinal flora results in the formation of DHD, and further reaction leading to the formation of unknown aliphatic compounds was implied (24).

Various bacterial phenotypes have been suggested to have a responsible role in daidzein metabolism in the small intestines of animals. An anaerobic bacterium, Clostridium sp. strain HGH6 (8), and a Clostridium-like strain, TM-40 (27), were found to reduce daidzein into DHD, and the C-ring cleavage was executed by a strain of Eubacterium (25). A human intestinal bacterium that could produce equol was first reported in 2005. Eggerthella strain Julong 732, which could not reduce daidzein into DHD, was found to reduce DHD into equol (28), thus establishing the aforementioned reduction sequence leading to the biologically active (S)-equol from daidzein via DHD in the human intestine (7, 10). Eggerthella species are normal residents of the human gut, and some species are implicated as causative agents of bacteremia (13). The microbial phenotypes that can reduce daidzein all the way into equol were recently isolated from mice (19), rats (20), pigs (33), and humans (18, 32). Nevertheless, the enzymology of the reduction, such as the nature of the enzyme responsible and the reaction mechanism, has yet to be established.

In the present study, the enzyme reaction mechanisms of two consecutive reduction reactions converting DHD into (3S)-equol were stereochemically assessed. To this end, four stereoisomers of THD were first synthesized, and their absolute configurations were determined. With the correlation of the absolute configuration of the synthetic THD isomers and the circular dichroism (CD) spectra at hand, the absolute configuration of THD produced through the cell-free bacterial reduction of DHD was determined. Each synthetic THD stereoisomer was then tested as a metabolic feedstock for (3S)-equol production during the growth of Julong 732. We found that only one of the THD steroisomers, (3R,4S)-THD, the very stereoisomer produced by the bacterial DHD reduction, was converted into (3S)-equol and that the final reduction accompanied the inversion of the configuration at C-3 of THD.

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Chemicals.
Daidzein and racemic equol were purchased from Indofine Co. (Somerville, NJ). Gifu anaerobic medium (GAM) was from Nissui Pharmaceutical Co. (Tokyo, Japan). Pd/C (10% Pd), sodium borohydride, and N,N-dimethyl formamide (DMF) were purchased from Aldrich (St. Louis, MO). Ammonium formate was obtained from Junsei Co. (Tokyo, Japan). Protease inhibitor cocktail, the reduced form of β-NADP (NADPH), and dithiothreitol were obtained from Sigma (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade solvents of acetonitrile, ethyl acetate, methanol, and acetic acids were obtained from Fisher (Pittsburg, PA).

General methods.
To monitor the progress of daidzein reduction, a Prostar HPLC system (Varian, Walnut Creek, CA) equipped with a photodiode array detector (Prostar 330; Varian) and a C₁₈ reversed-phase column (Spherisorb 5-µm ODS2, 4.6 by 250 mm; Waters, Clwyd, United Kingdom) was employed and monitored at 280 nm. 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 at 80:20 (vol/vol) for 3 min and linearly to 20:80 (vol/vol) for 12 min. The flow rate was 1 ml/min.

¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the compounds in dimethyl sulfoxide (d₆) were respectively obtained at 600 and 150 MHz on an Avance 600 NMR spectrometer (Bruker, Germany) at 296 K. The CD spectra of four THD stereoisomers in ethanol were measured using a J-715 CD spectropolarimeter (Jasco Corp., Tokyo, Japan). The spectra were recorded over the range of 200 to 310 nm using a cuvette with an optical path length of 0.5 cm.

Chemical synthesis and purification of DHD, THD, and DE.
DHD and THD were synthesized from daidzein through catalytic transfer hydrogenation employing ammonium formate and Pd/C as described previously by Wang et al. (28). The production of DHD and THD was monitored by direct injection into HPLC during the reaction. The reaction mixture was dried on a rotary vacuum evaporator after filtration and dissolved in DMF for HPLC analysis. DHD, cis-THD, and trans-THD were separated on the semipreparative C₁₈ reversed-phase column (Spherisorb S5 ODS2, 10 by 250 mm) under the same conditions described in the above section, except that the flow rate was 3 ml/min.

DE was obtained from the reduction of DHD by sodium borohydride. The reaction products were mainly THDs, as described by Joannou et al. (10), and a minute amount of DE was separated by the method described above.

Resolution of THD enantiomers.
A semipreparative chiral column (Sumichiral OA-7000, 8 by 250 mm; Sumika Chemicals, Osaka, Japan) was used to resolve cis- and trans-THDs. The mobile phase was composed of 100% H₂O (A) and 100% acetonitrile (B). The elution started with a linear gradient of an A/B ratio at 75:25 (vol/vol) to 80:20 (vol/vol) for 15 min and then moved linearly to 30:70 (vol/vol) for 10 min. Rechromatography of the collected fractions achieved complete resolution of four enantiomers from cis- and trans-THDs.

Bacterial culture and biotransformation.
The culturing of Julong 732 and the cell-free reaction were carried out in a Concept 400 anaerobic chamber (Ruskin Technology, Leeds, United Kingdom) under an atmosphere of 5% CO₂, 10% H₂, and 85% N₂. The stock of Eggerthella strain Julong 732 (KCCM 10490), preserved in liquid nitrogen, was thawed and incubated on a GAM agar plate for 4 days. A single colony was then transferred to the GAM broth. When the optical density at 600 nm (OD₆₀₀) reached 0.05 (typically 48 h after initiation of the culture), each THD substrate at 10 mM in DMF was added to the 500-µl culture to achieve a final concentration of 0.1 mM. After 16 h of incubation, when OD₆₀₀ reached 0.1, the culture was extracted with ethyl acetate, followed by evaporation to dryness in a vacuum centrifugal concentrator (Automatic Environmental SpeedVac; Thermo Savant, New York, NY) for 1.5 h. The dried extract was then dissolved in acetonitrile for HPLC analysis.

Cell-free incubation.
Two milliliters of the seed culture was added to 200 ml GAM broth and incubated for 1 day, after which 40 ml of the culture was inoculated into 800 ml GAM broth, and the bacteria were cultured for 8 h when the OD₆₀₀ reached 0.05. Crude DHD preparation was then added at the final concentration of 0.1 mM to induce the equol-producing enzyme(s). Bacteria were harvested when the OD₆₀₀ reached 0.1 in 16 h from 4.8 liters of the culture by centrifugation at 15,000 xg for 10 min at 4°C. The collected cells were resuspended in 5 volumes (wt/vol) of 50 mM sodium phosphate buffer, pH 7.2, containing 1 mM dithiothreitol and 1 mg protease inhibitor cocktail. The cells were then disrupted by sonication for 40 min on an ice bath. The disrupted cells were centrifuged at 15,000 xg for 30 min at 4°C, and the resulting supernatant was used for the incubation of the cell extract. The cell harvest and sonication were performed under aerobic conditions.

To start the reaction, 10 mM DHD in DMF and 50 mM NADPH in 50 mM sodium phosphate (pH 7.2) were added to the cell extract to achieve final concentrations of 0.1 mM DHD and 0.5 mM NADPH (26). After a 2-h reaction under anaerobic conditions, the reaction mixture was extracted with ethyl acetate. The dried ethyl acetate extract was dissolved in acetonitrile and injected into the semipreparative C₁₈ reversed-phase column (Waters Spherisorb S5 ODS2, 10 by 250 mm) to collect THD. The collected THD was resolved on a chiral column (Sumichiral OA-7000, 4.6 by 250 mm). The elution proceeded isocratically with an H₂O/acetonitrile ratio of 62:38 (vol/vol) for 60 min at 1 ml/min.

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Isolation and CD characteristics of THD stereoisomers.
The C₁₈ high-performance liquid chromatogram (Fig. 2) of the catalytic reduction products of daidzein showed the presence of DHD, trans-THD, and cis-THD, as well as minute amounts of equol and O-DMA. The isolated products were identified by comparison of their retention times, UV spectra, and NMR and mass spectra with the previously published data (28).

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FIG. 2. Reversed-phase HPLC profile of the products in catalytic hydrogenation of daidzein. The inset is the diode array trace of each peak. mAU, milliabsorbance units.

The trans- and cis-THDs, respectively, the racemic mixtures of (3R,4S)- and (3S,4R)-THDs and (3S,4S)- and (3R,4R)-THDs, were resolved into individual enantiomers by repetitive chiral column HPLC (Fig. 3). The enantiomers from trans-THD were temporarily designated as T1 and T2, and those from cis-THD as C1 and C2 (Fig. 3). Analysis by CD spectroscopy (Fig. 4) showed each stereoisomer exhibited a distinctive Cotton effect in the region of 200 to 300 nm (Fig. 4).

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FIG. 3. Separation of THD stereoisomers by chiral column HPLC. C1 and C2, enantiomers of cis-THD; T1 and T2, enantiomers of trans-THD. mAU, milliabsorbance units.

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FIG. 4. CD spectra of four THD stereoisomers. T1, (3R,4S); T2, (3S,4R); C1, (3S,4S); C2 (3R,4R).

Because no difference in the helicity-inducing chromophores was observed between isoflavan-4-ol and THD, the correlation of the absolute configuration with the Cotton effect in isoflavan-4-ol can be extrapolated to deduce the configuration of the THD isomers (31). (3R,4S)-Isoflavanol was shown to have both positive and negative Cotton effects in the 220- to 250-nm and 250- to 300-nm regions, respectively, and (3S,4S)-isoflavanol was shown to have two positive Cotton effects in the 220- to 250-nm region (31), whereas the enantiomers of both compounds have the opposite Cotton effects. Therefore, T1, which had positive and negative Cotton effects in the regions of 200 to 250 and 250 to 300 nm, respectively, reminiscent of the Cotton effect pattern of (3R,4S)- isoflavanol, was assigned to (3R,4S)-THD and T2 was identified as (3S,4R)-THD. C1 and C2 were assigned to (3S,4S)-THD and (3R,4R)-THD, respectively, in the same manner (Fig. 4).

Racemization of DHD by Julong 732.
Eight hours of incubation is necessary to achieve an equilibrium between (R)- and (S)-DHDs in the culture medium without cells under anaerobic conditions (29). However, the incubation of enatiomerically pure DHD in the cell extract and the whole cell accelerated the process to completion within 1 min and 3 h, respectively (data not shown). This rapid racemization, through keto-enol tautomerization, obscured the absolute configuration of the substrate DHD in the enzymatic reduction. Boiled cell extract did not catalyze this rapid racemization, thus supporting the biocatalytic nature of the acceleration.

Identification of the DHD reduction product.
Prior to the biotransformation experiments, possible nonenzymatic reductions of daidzein, DHD, and THD in GAM under the anaerobic atmosphere were examined. Under the experimental conditions, no reduction product was detected. Boiled cell extract under the anaerobic condition also could not reduce DHD into THD or equol.

The cell-free incubation of DHD was performed to collect THD, the intermediate of the DHD reduction. The separation of the reaction product by C₁₈ HPLC showed only the trans-THD fraction (Fig. 5, trace a, left panel), which was subjected to chiral column HPLC using authentic THDs, whose absolute configuration is known, as the standard. In the chiral HPLC of the isolated trans-THD fraction, T1 [(3R,4S)-THD] appeared as the sole product (Fig. 5, right panel). The accompanying small T2 peak was a carryover from the crude DHD preparation used in the enzyme induction, which contained T2 as a minor impurity (data not shown).

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FIG. 5. HPLC of the product(s) produced by cell-free incubation of DHD. (Left panel) C18 HPLC of cell-free incubation with DHD (trace a) and authentic sample (trace b). (Right panel) Chiral column HPLC of the separated trans-THD fraction (broken rectangle in the left panel): cell-free incubation with trans-THD (trace a) and authentic trans-THD (trace b). The small T2 peak on trace a of the right panel is due to T2 as an impurity in the DHD preparation used in enzyme induction. mAU, milliabsorbance units.

Biotransformation of THD by Julong 732.
(3S)-Equol was found in the Julong 732 culture incubated with either THD stereoisomer mixture (Fig. 6, left panel, trace a) or trans-THD (Fig. 6, left panel, trace b), whereas no metabolite was produced when cis-THD was fed (Fig. 6, left panel, trace c). Clearly, only T1 was preferentially removed with the concomitant formation of (3S)-equol in the culture (Fig. 6. left panel, trace b). To unequivocally confirm that T1 was the sole substrate of DHD reduction by Julong 732, incubation of each optically pure THD was monitored. Upon incubation of the individual stereoisomer of THD in the bacterial culture, only T1 was converted into (3S)-equol (Fig. 7c).

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FIG. 6. Chiral column HPLC of metabolite(s) produced by the strain Julong 732 fed with THD. (a) Mixture of THD stereoisomers; (b) trans-THD; (c) cis-THD. Ca, Cb, and Cc represent incubation in culture medium without cells. The T1, T2, C1, and C2 designations are the same as in Fig. 4. mAU, milliabsorbance units.

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FIG. 7. Chiral HPLC of metabolite(s) produced by Julong 732 fed with each THD stereoisomer. (a) (3S,4S)-THD (C1); (b) (3R,4R)-THD (C2); (c) (3R,4S)-THD (T1); (d) (3S,4R)-THD (T2). The T1, T2, C1, and C2 designations are the same as in Fig. 4. mAU, milliabsorbance units.

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Eggerthella strain Julong 732 metabolizes DHD into (3S)-equol. Because the intermediate DHD has two stereoisomers and the putative intermediate THD has four, the formation of (S)-equol from daidzein signifies the selection of one stereochemical course from the 16 stereochemical possibilities. To study the stereochemical preference of the DHD reduction leading to the formation of (3S)-equol, correlation between the absolute configuration and the spectroscopic and chromatographic behaviors of the authentic DHD and THD had to be established beforehand. Because the characteristics of the CD spectra of the DHD enantiomers are already known (29, 30), we synthesized four stereoisomers of THD and correlated the CD spectral characteristics with the absolute configurations of the isomers by applying the recently reported chiroptical properties of isoflavan-4-ols (31).

Several bacteria—a bovine rumen anaerobe, Lactobacillus sp. strain Niu-O16 (29); a human intestinal Clostridium sp. strain, HGH6 (8); and a Clostridium-like bacterium strain, TM-40 (27), to name a few—were reported to reduce daidzein into DHD. Wang et al. (29) attempted to establish the stereochemistry of daidzein reduction into DHD by strain Niu-O16. However, tautomerization-assisted equilibrium between (R)-DHD and (S)-DHD prevented them from determining the chirality at C-3 of DHD as the product (29). This tautomerization would also make it impractical to determine the substrate stereospecificity of the proposed DHD reductase of Julong 732. Nevertheless, Wang et al. argued that DHD reduction and the following THD reduction by Julong 732 proceed in a stereospecific manner to retain the configuration at C-3 (28). Based on their proposal, we, at first, hypothesized that THD, as the immediate precursor of (3S)-equol, would have the 3S configuration, with the configuration at C-4 unknown. DHD as the substrate of the putative DHD reductase would also have the 3S configuration.

Because DHD enantiomers are in equilibrium through a slow racemization in an aqueous environment to allow the resolution into each enantiomer (29), we reasoned that the use of a single DHD enantiomer in the cell-free biotransformation system could differentiate the stereospecificity of the putative DHD reductase of Julong 732, under the assumption that the reduction proceeds faster than the racemization. However, the unexpected acceleration of the racemization in the presence of either the cell extract or the whole cell obscured the substrate specificity of the DHD reduction. However, the DHD racemase activity of Julong 732 has one beneficial consequence: it allows this strain to completely convert DHD, regardless of its C-3 configuration, into THD.

The intermediacy of THD in the (3S)-equol production from DHD by Julong 732 was established by exploiting the cell-free biotransformation of DHD. The incubation of DHD in the cell extract allowed the accumulation of a sizable amount of THD (Fig. 5), whereas the accumulation of THD could not be attained through the whole-cell incubation. It should be noted that preincubation of the bacterial culture with DHD was necessary for the preparation of an active cell extract; the extract prepared from the untreated cells did not have the DHD-reducing activity. This alluded that the DHD-reducing enzyme of Julong 732 was inducible. Recently, a strain of Coriobacteriaceae, to which the genus Eggerthella belongs, was described to have inducible daidzein-reducing enzymes (19).

Now, with the absolute configuration of the product THD at hand as (3R,4S), we are able to propose a possible reduction mechanism involving the tautomerization of DHD (Fig. 8). In this mechanism, (3S)-DHD would be in equilibrium with (3R)-DHD through tautomerization with the intermediate enol 1 (Fig. 8, pathway A), which was subsequently reduced into THD. This putative mechanism could explain that the absolute configuration at C-3 of the intermediate THD is R (see below). A simple reduction of the C-4 carbonyl group could be an alternative (Fig. 8, pathway B), although this would require a separate racemization mechanism.

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FIG. 8. Proposed reduction/racemization mechanism of the putative DHD reductase.

The fact that the absolute configuration of the putative DHD reductase product was (3R,4S)-THD does not necessarily mean that this stereoisomer was a direct substrate of the THD reductase. For example, racemization of (3R)-THD into (3S)-THD, with a subsequent simple reduction, by two separate enzymes could be considered. We thus proceeded to determine the substrate specificity of the THD reduction with each of the four THD stereoisomers to affirm that the product of the first reduction, (3R,4S)-THD, was the true substrate of the next reduction step to produce (3S)-equol. We confirmed that indeed Julong 732 stereospecifically reduced (3R,4S)-THD into (3S)-equol (Fig. 1).

The fact that (3R,4S)-THD was the intermediate of DHD reduction into (3S)-equol contradicted our initial assumption that the configuration at C-3 would not change during the reduction process (28). Rather, we found that the reduction of (3R,4S)-THD into (3S)-equol was accompanied by the inversion of the configuration at C-3. This reduction can be depicted as the sum of two consecutive reactions: carbon-carbon double-bond reduction after dehydration, in analogy to the reactions by 3-hydroxy-acyl carrier protein reductase (17), followed by enoyl-acyl carrier protein reductase (22) in the fatty acid biosynthesis. However, the action of dehydratase on THD would result in the production of DE, which was not the precursor of (3S)-equol in the whole-cell incubation experiment (data not shown). Therefore, the mechanism involving DE as an intermediate was discarded. One of the possible explanations for this peculiar stereochemical outcome is depicted in Fig. 9. In the mechanism depicted in Fig. 9, the carbocation 2, formed as a result of the removal of a hydroxyl group from C-4, would then undergo a conformation change to give rise to the conformer 3, so that the suprafacial 1,2-hydride migration of H-3 into the C-4 axial position could occur. The hydride transfer to re-face at C-3 of the intermediate 4 would complete the reduction with the inversion of the configuration to yield (3S)-equol. The mechanisms proposed in Fig. 9 could be tested using (3R,4S)-THD labeled with a deuterium at C-3.

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FIG. 9. Proposed mechanism of the putative THD reductase of Julong 732.

If THD is a structurally imminent intermediate of (3S)-equol, the production of (3S)-equol from THD should be more efficient than that from DHD. Although Wang et al. (28) reported that THD was a poor precursor for (3S)-equol production compared to DHD in a whole-cell system, we observed a rapid cell-free formation of (3S)-equol from T1, namely (3R,4S)-THD, in the presence of NADPH, which was found to be a better hydride donor than NADH (26). Maximum production of (3S)-equol was observed in about 1 h with THD, whereas DHD required at least 3 h to achieve maximal production (data not shown).

In summary, we first established the configuration of four THD stereoisomers and correlated their absolute configurations with the behavior in the chiral column chromatography. With these data at hand, the stereochemical course of DHD reduction into (3S)-equol via THD by the human intestinal bacterium Eggerthella strain Julong 732 was determined. (3R)-DHD was reduced to (3R,4S)-THD, which was further reduced to (3S)-equol. The equol formation was accompanied by the inversion of the configuration at C-3 of (3R,4S)-THD. The present experiment firmly established the sole intermediacy of THD in the DHD reduction into equol, at least by Julong 732.

 
We thank the Ministry of Education, Science and Technology, through the BK21 Program administered by the Department of Agricultural Biotechnology, Seoul National University, for support. J.H. thanks the Korea Research Foundation (grant KRF-2008-code-F00014), funded by the Korean Government, for support.

We appreciate the critical review of the manuscript by Hor-Gil Hur at the Gwangju Institute of Science and Technology.

 
* Corresponding author. Mailing address: Program in Applied Life Chemistry, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-921, South Korea. Phone: 82-2-880-4642. Fax: 82-2-873-3112. E-mail: soounkim{at}plaza.snu.ac.kr

Published ahead of print on 20 March 2009.

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Applied and Environmental Microbiology, May 2009, p. 3062-3068, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02058-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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