Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

ABSTRACT

The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (−)-taxifolin. The K_m for (+)-taxifolin was 6.4 ± 0.8 μM, and the V_max was 108 ± 4 μmol min⁻¹ (mg protein)⁻¹. The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus. Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439–1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus.

IMPORTANCE Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus. Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

INTRODUCTION

Flavonoids represent a major group of plant-derived polyphenolic compounds and have been implicated in beneficial effects on human health (1–4). The flavonoids are classified into several subgroups, which include, among others, flavonols, flavanonols, flavones, flavanones, and chalcones. Quercetin (Fig. 1) is a highly abundant dietary flavonoid that shows a broad range of biological activities. Thus, this flavonol has been intensively studied with respect to its role in disease prevention and use in therapy (5–7). The effects of quercetin and other flavonoids depend on how they are metabolized in the human body, including their conversion by intestinal bacteria (8). Beside deglycosylation and deconjugation, intestinal bacteria are also able to further transform the resulting flavonoid aglycones. However, knowledge about the corresponding bacterial species and, in particular, the enzymes involved is still limited.

FIG 1.

Compounds used in this study.

Eubacterium ramulus and Flavonifractor plautii (formerly Clostridium orbiscindens) are human intestinal bacteria that have been demonstrated to cleave the central heterocyclic C-ring of flavonols and flavones (9–12). Both species are strict anaerobes and common inhabitants of the human intestine (12, 13). The pathways of anaerobic degradation of flavonols and flavones (see Fig. S1 in the supplemental material) have been found to be identical in both E. ramulus and F. plautii (11, 12). The degradation of the flavonol quercetin starts with the reduction of the double bond in the 2,3-position of the C-ring, resulting in the formation of the flavanonol taxifolin (trans-dihydroquercetin). Taxifolin is isomerized to the auronol alphitonin by a unique C-ring contraction (structures in Fig. 1; also see Fig. S1). Alphitonin is further converted by oxidative decarboxylation to 3,4-dihydroxyphenylacetic acid and phloroglucinol, the latter undergoing further degradation to butyrate and acetate (11, 12). The degradation of flavones starts with a reduction step analogous to that of flavonols resulting in the formation of the corresponding flavanones (see Fig. S1). In contrast to flavonol degradation, further steps in flavanone degradation encompass the C-ring fission to a chalcone, reduction to a dihydrochalcone, and hydrolysis to the corresponding hydroxyphenylpropionic acid and phloroglucinol (11, 12).

Three enzymes from E. ramulus involved in the degradation of flavones or flavanones have been characterized so far, and the corresponding genes have been identified: chalcone isomerase (CHI), enoate reductase (ERED), and phloretin hydrolase (Phy) (14–16). These enzymes catalyze the conversion of flavanones (e.g., naringenin [Fig. 1]) via chalcones (e.g., naringenin chalcone [Fig. 1]) and dihydrochalcones (e.g., phloretin) to monophenolic metabolites [e.g., 3-(4-hydroxyphenyl)-propionic acid and phloroglucinol] (see Fig. S1 in the supplemental material). The structures of CHI and Phy have recently been solved, and catalytic amino acid residues have been identified by mutagenesis (15, 17, 18). CHI was crystallized in both ligand-free and ligand-bound forms, and the interaction of the active site His33 with naringenin was elucidated (15, 17).

The key activity in the pathway of flavonol/flavanonol conversion, the taxifolin isomerization, has been detected not only in intact cells but also in cell-free preparations of E. ramulus, including a partially purified protein fraction (11). The kinetics of the nonenzymatic thermal rearrangement of taxifolin to alphitonin has been investigated, which is considerably less efficient than the enzyme-catalyzed isomerization (19). The present study aimed at the purification and characterization of the taxifolin-isomerizing enzyme from E. ramulus, including the identification, cloning, and heterologous expression of the encoding gene.

MATERIALS AND METHODS

Chemicals.

(±)-Taxifolin and (±)-trans-dihydrokaempferol were purchased from Sigma (Deisenhofen, Germany). (+)-Taxifolin, butein, naringenin, eriodictyol, quercetin, phloretin, and catechin were from Roth (Karlsruhe, Germany). Naringenin and eriodictyol were identified as racemic compounds by chiral high-performance liquid chromatography (HPLC) analysis. (+)-Dihydrokaempferol (aromadendrin) was from Arbonova (Turku, Finland). Alphitonin was obtained by enzymatic transformation of taxifolin followed by purification and structure elucidation by nuclear magnetic resonance (NMR) analysis (11). (±)-Flavanonol was obtained by oxidation of the corresponding chalcone (1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one) using hydrogen peroxide (20).

Bacterial strains, growth conditions, and preparation of cell extracts.

Eubacterium ramulus DSM 16296 was grown anoxically in a complex medium (ST medium) as described previously (11). Cell extracts were prepared under air using a French pressure cell (Aminco, Silver Spring, MD) as described elsewhere (11). Flavonifractor plautii DSM 4000^T was grown anoxically at 37°C in brain heart infusion (BHI) broth (Roth, Karlsruhe, Germany) supplemented with 3 mM cysteine and using a gas phase of 80:20 (vol/vol) H₂-CO₂.

Transformed Escherichia coli JM109 and KRX cells (Promega) were grown oxically in LB broth (Lennox, Roth) supplemented with carbenicillin (100 μg ml⁻¹ [Roth]), with shaking (250 rpm) at 37°C unless indicated otherwise. For preparation of cell extracts, bacteria were harvested by centrifugation (10,000 × g, 10 min, 4°C), washed with 50 mM potassium phosphate buffer (PPB) (pH 6.8) and subsequently suspended in the same buffer. Cells were disrupted by shaking with 0.1 mm zirconia-silica beads (Roth) for 20 s at 6.0 m s⁻¹ in a FastPrep instrument (FP120 system; MP Biomedicals, Heidelberg, Germany). Cell debris and unbroken cells were sedimented by centrifugation (12,000 × g, 20 min, 4°C). Cell growth was monitored by measuring the attenuance at 600 nm. The protein concentration of cell extracts was determined by the Bio-Rad Bradford assay using bovine serum albumin as a standard.

Purification of the taxifolin-isomerizing enzyme from E. ramulus.

Enzyme purification was performed at 4°C using a Pharmacia LKB fast-performance liquid chromatography (FPLC) system. The cytosolic fraction (ca. 300 mg protein) obtained by ultracentrifugation (110,000 × g, 4°C, 1 h) of the cell extract was loaded onto a DEAE-Sephacel (Amersham Pharmacia Biotech, Freiburg, Germany) column (6 by 2.5 cm) equilibrated with 50 mM PPB (pH 6.8). Elution was done with a linear gradient of KCl (0 to 1 M) in 50 mM PPB (pH 6.8) at a flow rate of 2 ml min⁻¹. Fractions with taxifolin-isomerizing activity were pooled, and (NH₄)₂SO₄ was added to a final concentration of 1 M. The protein solution was loaded onto a HiLoad phenyl Sepharose HP 16/10 column (Amersham Pharmacia Biotech) equilibrated with 1 M (NH₄)₂SO₄ in 50 mM PPB (pH 6.8). Elution was done with a linear gradient of (NH₄)₂SO₄ (1 to 0 M) in 50 mM PPB (pH 6.8) at a flow rate of 1 ml min⁻¹. Pooled active fractions were concentrated and desalted (Centriprep 10 cartridge; Amicon) and loaded onto a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) equilibrated with 50 mM PPB (pH 6.8). Elution was done with a linear gradient of KCl (0 to 1 M) in 50 mM PPB (pH 6.8) at a flow rate of 1 ml min⁻¹. Active fractions were pooled, concentrated (Centricon 10 cartridge; Amicon), and applied to a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). Elution was done with 50 mM PPB (pH 6.8) supplemented with 100 mM KCl at a flow rate of 0.25 ml min⁻¹. To the pooled active fractions, (NH₄)₂SO₄ was added to a final concentration of 1 M. The protein solution was loaded onto a phenyl Superose HR 5/5 column (Amersham Pharmacia Biotech) equilibrated with 1 M (NH₄)₂SO₄ in 50 mM PPB (pH 6.8). Elution was done with a linear gradient of (NH₄)₂SO₄ (1 to 0 M) in 50 mM PPB (pH 6.8) at a flow rate of 1 ml min⁻¹, and active fractions were pooled.

Enzyme assays.

The assay mixtures contained 50 mM PPB (pH 6.8), 60 to 600 μM (+)-taxifolin (stock solutions in dimethyl sulfoxide [DMSO]; final DMSO concentration, 5%), and an enzyme source (final protein concentration, 0.1 to 200 μg ml⁻¹). The assay mixture was incubated at 23°C. Samples were withdrawn at different time points and mixed with 1 volume of methanol-water-acetic acid (50:45:5 [vol/vol/vol]) to stop the reaction. After centrifugation (12,000 × g, 5 min), an aliquot (5 to 60 μl) of the supernatant was analyzed by reversed-phase HPLC (RP-HPLC), as detailed below.

In addition to the assay described above, the following modifications were tested. The incubation temperature varied in the range of 15 to 45°C (steps of 2°C), and the pH ranged from 5.2 to 8.0 (steps of 0.2 pH unit). (±)-Taxifolin, (+)-dihydrokaempferol, (±)-dihydrokaempferol, flavanonol, butein, phloretin, quercetin, catechin, naringenin, and eriodictyol were tested as the substrates. For inhibition experiments, the following flavonoids were combined in one assay each: (+)-taxifolin (60 μM) and flavanonol (60 or 600 μM), (+)-taxifolin (180 μM) and eriodictyol (180 μM), and (+)-dihydrokaempferol (180 μM) and naringenin (180 μM). For chiral analyses, stock solutions in 70% (vol/vol) methanol of (+)-taxifolin, (+)-dihydrokaempferol, (±)-taxifolin, and (±)-dihydrokaempferol were applied to final concentrations of 100 μM (enantiomers) or 200 μM (racemates). Aliquots (5 μl) from these assays were analyzed by chiral HPLC, as detailed below.

Enzyme activities were calculated based on the product formation using nonlinear regression of data points (GraFit 5; Erithacus Software, Surrey, United Kingdom). Initial rates, v, were determined by using the equation [P] = v (1 − e^−kt) k⁻¹, where [P] is the product concentration at time t and k is the corresponding first-order rate constant for the gradual deceleration of the reaction. Initial rates for butein isomerization were determined based on the substrate consumption by using the equations [S] = [S₀] e^−kt and v = k [S₀], where [S] is the substrate concentration at time t and [S₀] is the initial substrate concentration. The enzymatic activities were calculated by subtracting the rate of spontaneous butein cyclization determined in the absence of the enzyme. The reported standard errors of the rates relate to the nonlinear regression of at least four data pairs.

HPLC analyses.

Flavonoids were analyzed by RP-HPLC as described previously (11). Samples obtained in the course of taxifolin conversion by recombinant CHI were analyzed using aqueous 2% acetic acid (solvent A) and methanol (solvent B) with a modified gradient (B from 5 to 95% in 12 min).

Chiral HPLC analysis was performed as previously described (21) with the following modifications. Water acidified to pH 3 with trifluoroacetic acid (solvent A) and acetonitrile (solvent B) served as the mobile phase in a gradient mode (B at 10%, held for 5 min, from 10 to 20% in 25 min, from 20 to 70% in 10 min) at a flow rate of 0.5 ml min⁻¹. Detection was at 290 nm; UV spectra were recorded in the range of 200 to 400 nm.

Calibration curves of the corresponding reference compounds were used for quantification. Maesopsin concentrations were determined applying the calibration curve of alphitonin.

SDS-PAGE.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done according to Laemmli (22) using 13% resolving gels combined with 5% stacking gels. Unstained marker proteins (LMW electrophoresis calibration kit; GE Healthcare) or prestained marker proteins (PageRuler Plus prestained protein ladder; Fermentas, St. Leon-Rot, Germany) were used as molecular mass standards. Protein bands were stained with Coomassie brilliant blue R-250 (0.2% [wt/vol] in 45:45:10 [vol/vol/vol] methanol-acetic acid-water).

Determination of native molecular mass.

Native molecular masses were estimated by gel filtration and native PAGE, respectively. Gel filtration of the taxifolin-isomerizing enzyme was carried out on a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) using a Pharmacia LKB FPLC system. Elution was done with 50 mM PPB (pH 6.8) supplemented with 100 mM KCl at a flow rate of 0.25 ml min⁻¹ for the native enzyme and with 50 mM PPB (pH 6.8) supplemented with 150 mM KCl at a flow rate of 0.5 ml min⁻¹ for the recombinant enzyme. Molecular mass standards (HMW and LMW gel filtration calibration kit; GE Healthcare) included ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa).

For native PAGE, precast gels with a linear gradient of 4 to 20% acrylamide (Ready Gel, Tris-HCl; Bio-Rad) were used. Gels were run in Tris-glycine buffer (10 mM Tris, 77 mM glycine [pH 8.5]) at 150 V. Bio-Rad precision protein standards were used as marker proteins. Protein bands were stained either with Coomassie brilliant blue R-250 as described above or with the Bio-Rad silver stain kit.

Preparation of genomic DNA from bacteria.

E. ramulus cells from a 50-ml overnight culture were pelleted by centrifugation (3,000 × g, 4°C, 15 min), resuspended in 5 ml of 50 mM Tris-HCl (pH 8.0) containing 25% (wt/vol) sucrose, and incubated at 22°C for 30 min. The cells were pelleted and resuspended in 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and then frozen at −80°C for 1.5 h followed by a heat shock at 50°C for 5 min. Lysozyme (0.7 ml, 1 mg ml⁻¹) was added, and the suspension was incubated overnight at 4°C. After addition of 1 ml of STEP solution (50 mM Tris-HCl [pH 7.5], 0.4 M EDTA, 0.5% [wt/vol] SDS) and 1 mg of proteinase K, the mixture was incubated at 50°C for 1 h followed by treatment with 4 μl of DNase-free RNase (12 mg ml⁻¹) at 22°C for 10 min. Cell lysates were successively extracted with 1 volume each of Tris-saturated phenol (Biomol, Hamburg, Germany), Tris-buffered phenol–chloroform–isoamyl alcohol (24:24:1 [vol/vol/vol]), and chloroform. DNA was precipitated with 1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of 96% (vol/vol) ethanol, washed with 70% (vol/vol) ethanol, dried, and dissolved in TE buffer. Genomic DNA from F. plautii was isolated with the RTP Bacteria DNA minikit (Invitek, Berlin, Germany).

Cloning of the gene encoding the taxifolin-isomerizing enzyme and flanking genes.

The protein band representing the taxifolin-isomerizing enzyme was excised from the gel following SDS-PAGE with the purified enzyme. Tryptic in-gel digestion of the protein, mass spectrometric analysis of the resulting peptides, and de novo sequencing were performed as described previously (23).

PCR with the degenerate primers CHI 2f and TI 6r (see Table S1 in the supplemental material) deduced from the N-terminal sequence of CHI from E. ramulus (14) and from a peptide sequence of the taxifolin-isomerizing enzyme, respectively, led to the amplification of a 522-bp PCR product. The PCR mixture (25 μl) contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM each deoxynucleoside triphosphate (dNTP), 4 μM each primer, 1.25 U Taq DNA polymerase (Invitrogen), and 0.2 μl genomic DNA solution. The PCR program was as follows: 94°C for 5 min, 30 cycles of 94°C for 1 min, 53.4°C for 1 min, 72°C for 1.5 min, and finally 72°C for 5 min. The PCR product was purified and cloned into the pGEM-T Easy vector followed by transformation of E. coli JM109 with the resulting plasmid (pGEM-T Easy vector system; Promega). Plasmid DNA was isolated from positive transformants and sent out for sequencing of the cloned insert. Based on the initially amplified DNA region, complete sequences of the gene of interest and the flanking genes were obtained by inverse PCR amplifications with self-ligated genomic DNA libraries as described previously (21) using the restriction enzymes BamHI and PstI (New England BioLabs) and the primers TI 7r and TI 8f (see Table S1). The resulting PCR products were cloned as described above and sequenced.

Sequence analyses.

DNA sequencing was carried out by Eurofins MWG Operon (Ebersberg, Germany). The Vector NTI Suite 9 software package (Invitrogen) was employed to process and assemble the sequenced DNA fragments. Sequence similarity search was done with the Basic Local Alignment Search Tool (BLAST; www.ncbi.nlm.nih.gov/blast). Search for putative transcription promoter and terminator sequences was done by the web-based programs BPROM and FindTerm, respectively (www.softberry.com). For prediction of the protein localization and the membrane protein topology, the web-based program PSORT (www.psort.org) was applied.

Heterologous gene expression in E. coli and purification of tagged proteins.

For expression of CHI from E. ramulus, the encoding gene and adjacent potential regulatory sequences were amplified by PCR as described above using the primers TI 9f and TI 10r (see Table S1 in the supplemental material) and 0.5 µl of genomic DNA from E. ramulus (diluted 1:100 with water) as a template. The annealing temperature of 68°C was applied for 30 s. The obtained PCR product was purified and ligated into the pGEM-T Easy vector, followed by transformation of E. coli JM109 with the resulting plasmids as described above. The expression of CHI was tested in the absence and presence of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG [1 mM]), and samples were withdrawn after 4, 8, and 20 h for preparation of cell extracts as described above.

The gene from F. plautii was cloned by genetically fusing Strep-tag II to its N or C terminus using the StarGate cloning system (IBA, Goettingen, Germany). The PCR mixture (25 μl) for gene amplification contained 1× Q5 high-fidelity master mix (New England BioLabs), 0.5 μM primers FP-CF and FP-CR (see Table S1 in the supplemental material), and 50 ng of genomic DNA. The PCR program was as follows: 98°C for 30 s, 25 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 1 min and finally 72°C for 2 min. The gene cloned in pPSG-IBA 3 or pPSG-IBA 5 was expressed in E. coli KRX following induction by rhamnose (0.1% [wt/vol]) and cultivation overnight at 25°C. Purification of tagged proteins from cell extracts was done using a 1-ml Strep-Tactin Sepharose column (IBA) as described previously (21).

Preparation of maesopsin.

(+)-Dihydrokaempferol (2.9 mg [final concentration, 10.07 mM], 5% DMSO) was incubated in 50 mM PPB (pH 6.8) with the cell extract of chi-expressing E. coli pTI2 (final protein concentration, 9 μg ml⁻¹) at 37°C for 6 h with shaking. After centrifugation at 18,000 × g for 5 min, 30-μl samples were applied to RP-HPLC. The fractions containing the product of dihydrokaempferol conversion were manually collected, pooled, and dried by vacuum centrifugation (RC 10.22; Jouan, Saint-Nazaire, France).

¹H NMR spectra (500 MHz) and ¹³C NMR spectra (125 MHz) were recorded in DMSO-d₆ using a Bruker Avance 500 instrument: ¹H NMR δ 2.86 (d, 1H, J = 13.85 Hz, CHH), 2.91 (d, 1H, J = 13.85 Hz, CHH), 5.68 (s, 2H, 7-H), 5.73 (s, 2H, 5-H), 6.52 (d, 2H, J = 8.5 Hz, 3′-H), 6.90 (d, 2H, J = 8.5 Hz, 2′-H); ¹³C NMR δ 40.76 (CH₂), 89.73 (C-7), 95.87 (C-5), 101.34 (C-2), 105.66 (C-3a), 114.69 (C-3′), 124.48 (C-1′), 131.37 (C-2′), 155.94 (C-4′), 158.15 (C-4), 168.34 (C-6), 171.87 (C-7a), 192.94 (CO). The data were in agreement with those determined previously for maesopsin (24).

Molecular docking.

The X-ray crystal structures of CHI from E. ramulus (PDB ID no. 3ZPH and 4D06) (15, 17) were obtained from the RCSB Protein Data Bank (www.rcsb.org). The structures were used as a template for flexible ligand docking with AutoDock 4.2 (25). Prior to the docking calculations, crystallographic water molecules were removed from the template structure and hydrogen atoms were added using Molecular Operating Environment (MOE 2014.09; Chemical Computing Group, Montreal, Canada). Atomic partial charges were calculated using AutoDock Tools (25). (+)-Taxifolin and (−)-taxifolin were docked into the putative active site of the enzyme. The active-site area was defined around the binding pockets in the proximity of residues Asp79 and Gln101 as proposed by Gall et al. (15). Putative compound binding modes were selected after visual inspection of high-scoring docking poses.

Site-directed mutagenesis.

The mutation was introduced by PCR with the forward primer TI 13f carrying the desired nucleotide changes and the reverse primer TI 14r (see Table S1 in the supplemental material) using the Q5 site-directed mutagenesis kit (New England BioLabs). The chi-containing plasmid pTI2 served as a template. E. coli JM109 was transformed as described above with the resulting plasmid carrying the mutated chi gene. The cloned gene was sequenced to verify the introduction of the desired mutation.

Accession number(s).

The sequence encompassing chi and adjacent genes from E. ramulus has been deposited in the GenBank database under accession no. KM067454.

RESULTS

Purification of the taxifolin-isomerizing enzyme from E. ramulus and characterization of the purified enzyme.

The enzyme isomerizing taxifolin to alphitonin was purified from cell extracts of E. ramulus to apparent homogeneity using a five-step protocol. Starting from the cytosolic fraction, the overall purification was up to 610-fold with a yield of 10% (Table 1). SDS-PAGE analysis of the purified enzyme revealed a single protein band of 31 kDa (see Fig. S2A in the supplemental material). Analysis under nondenaturing conditions by native PAGE showed a main protein band at 120 kDa and a faint band at 230 kDa (see Fig. S2B). The apparent molecular mass determined by gel filtration was 260 kDa. These results suggested that the protein exists as a homotetramer or a homo-octamer. The purified enzyme catalyzed the isomerization of (+)-taxifolin to alphitonin in the absence of any external coenzyme at a specific activity of 108 ± 4 μmol min⁻¹ (mg protein)⁻¹. The K_m for (+)-taxifolin was 6.4 ± 0.8 μM. The resulting k_cat (calculated per monomer) and k_cat/K_m were 58 ± 2.2 s⁻¹ and 9.1 × 10⁶ M⁻¹ s⁻¹, respectively. The temperature optimum of the taxifolin isomerization was 39°C, whereas the pH was optimal between 5.2 and 7.0.

TABLE 1.

Purification steps for the taxifolin-isomerizing enzyme from E. ramulus

Step	Total protein (mg)	Total activity (U)	Sp act (U mg protein−1)	Purification (fold)	Yield (%)
Cytosolic fraction	262	48	0.18	1.0	100
DEAE-Sephacel	53.8	34	0.63	3.5	71
Phenyl Sepharose	4.34	29	6.7	37	61
Mono Q	1.28	18	14	77	38
Superdex 200	0.34	11	33	180	23
Phenyl Superose	0.04	4.9	110	610	10

The taxifolin-isomerizing enzyme completely transformed (+)-(2R,3R)-taxifolin to equimolar amounts of alphitonin but only 50% of the racemic (±)-taxifolin. Chiral HPLC analysis following incubation of the enzyme with (±)-taxifolin confirmed its stereospecificity (Fig. 2). Only (+)-taxifolin (retention time [t_R] = 19.6 min) was accepted as a substrate and converted to alphitonin, which showed one relatively broad peak (t_R = 8.9 min). The initial conformation of the produced alphitonin, a compound with one chiral C-atom, could not be clarified due to the fast on-column racemization of this auronol (19). The corresponding enantiomer (−)-(2S,3S)-taxifolin (t_R = 19.1 min) was not converted and, moreover, reduced the isomerization rate of (+)-taxifolin to 58% when present at the identical concentration. The cis isomers (+)-epitaxifolin (t_R = 18.7 min) and (−)-epitaxifolin (t_R = 20.1 min), which were contained in the starting material as minor components and whose assignment was based on circular dichroism (19), were not converted either (Fig. 2).

FIG 2.

Stereospecific isomerization of flavanonols by the taxifolin-isomerizing enzyme subsequently identified as CHI. Racemic mixtures of the flavanonols were incubated with the enzyme purified from E. ramulus, and samples were analyzed by chiral HPLC. Incubation of (±)-taxifolin (a) led to the conversion of only (+)-taxifolin to (±)-alphitonin (b). Incubation of (±)-dihydrokaempferol (c) led to the conversion of only (+)-dihydrokaempferol to (±)-maesopsin (d). mAU, milliabsorbance units.

The taxifolin-isomerizing enzyme also converted the related flavanonol dihydrokaempferol (structure in Fig. 1), which lacks the 3′-hydroxy group of taxifolin. As observed for taxifolin, exclusively (+)-(2R,3R)-dihydrokaempferol (t_R = 23.6 min) served as a substrate but not the corresponding (−)-(2S,3S)-enantiomer (t_R = 23.2 min) (Fig. 2). The product formed was identified as (±)-maesopsin (structure in Fig. 1) by NMR analysis following its purification. As observed for alphitonin, maesopsin exhibits only one peak (t_R = 15.4 min) in chiral HPLC analysis (Fig. 2), which was attributed again to a fast on-column racemization. The conversion of (+)-dihydrokaempferol proceeded at 55% of the rate observed for (+)-taxifolin. In the presence of an equimolar concentration of (−)-dihydrokaempferol, the isomerization rate of (+)-dihydrokaempferol decreased to 60%.

(±)-Flavanonol with unsubstituted A- and B-rings (structure in Fig. 1), which represents the basic structure of this flavonoid class, was not transformed by the taxifolin-isomerizing enzyme. This compound did not inhibit the taxifolin conversion, at neither the same nor a 10-fold higher concentration. Among members of other flavonoid classes, only the chalcone butein (structure in Fig. 1) served as a substrate for the taxifolin-isomerizing enzyme, while conversion of quercetin, naringenin, eriodictyol (structures in Fig. 1), phloretin, and catechin was not observed. Butein was cyclized to the corresponding flavanone, however, at a rate 2 orders of magnitude lower (<1% activity) compared with taxifolin isomerization.

Identification of the gene encoding the taxifolin-isomerizing enzyme.

Several oligonucleotides deduced from appropriate peptide sequences of the tryptically digested taxifolin-isomerizing enzyme were used as primers in PCR to amplify the corresponding gene fragments. In addition, one primer designed based on the previously determined N terminus of CHI from E. ramulus (14) was included based on the assumption that the genes encoding flavonoid-converting enzymes neighbor each other on the genome. The only PCR product was obtained by combining the CHI-derived primer with one primer based on a peptide sequence of the taxifolin-isomerizing enzyme. This amplicon exhibited an unexpectedly small size of ca. 500 bp. By amplification of adjacent DNA regions by inverse PCR, the complete gene sequence was obtained. It comprises 849 bp encoding a protein of 283 amino acids. The calculated molecular mass of 32,448 Da was in accordance with that observed for the taxifolin-isomerizing enzyme by SDS-PAGE. The predicted cytosolic localization corresponds to the recovery of the taxifolin-isomerizing activity in the cytosolic and not the membrane fraction. The deduced amino acid sequences matched several peptide sequences of the taxifolin-isomerizing enzyme determined by mass spectrometric analysis (see Fig. S3 in the supplemental material), confirming the identity of its encoding gene. The N terminus was found to be largely identical to that determined for the CHI from E. ramulus (14). It finally turned out that the complete gene sequence of the taxifolin-isomerizing enzyme is identical to that of the CHI-encoding gene of E. ramulus DSM 16296 (GenBank accession no. KF154734), which has been published recently (15). The corresponding amino acid sequence of CHI (accession no. AGS82960) also showed 99% identity with a hypothetical protein (accession no. ERK52514) from the completely sequenced E. ramulus type strain ATCC 29099 (see Fig. S4 in the supplemental material). Further database search revealed similarity to putative acetyl coenzyme A (acetyl-CoA) hydrolases from several bacteria, including Clostridium sp. strain SY8519 (68% identity; accession no. BAK48446), and to a hypothetical protein from F. plautii (50% identity; accession no. EHM54434) (see Fig. S4).

Amplification and sequencing of the DNA regions adjacent to the CHI gene (chi) from E. ramulus DSM 16296 resulted in identification of three potential gene sequences (usg, dsg1, and dsg2), all oriented in the same direction as chi (see Fig. S5 in the supplemental material). The sequence data, including the gene cluster (3,590 bp), have been submitted to the GenBank database under accession no. KM067454. The potential gene upstream of chi (positions 1180 to 2028), referred to as usg, comprises 975 bp (positions 114 to 1088). The corresponding gene product of 325 amino acids is a putative flavin reductase characterized by an N-terminal domain of NADPH-dependent flavin mononucleotide (FMN) reductases, including a cofactor binding site (GXXXGXG; amino acid positions 55 to 61) and an Fe/S cluster binding motif (CXXCXXC; amino acid positions 46 to 52). The two genes downstream of chi, referred to as dsg1 (483 bp; positions 2278 to 2760) and dsg2 (partial sequence, 728 bp; positions 2863 to 3590) most likely encode a MoaB/MogA-like molybdopterin biosynthesis enzyme and a MoeA-like molybdopterin biosynthesis enzyme, respectively. Hypothetical proteins with 99% identity to Usg, Dsg1, and Dsg2 are encoded by genes likewise situated upstream and downstream, respectively, of the gene corresponding to chi in the E. ramulus type strain, ATCC 29099. These include a putative flavin reductase (ERK52513), the MoaB/MogA family molybdenum cofactor synthesis domain protein (ERK52516), and an MoeA-like molybdopterin binding domain protein (ERK52517).

Of the eight predicted promoter sequences, the three most highly ranked ones are present upstream of usg (positions 45 to 71), chi (positions 1116 to 1146), and dsg1 (positions 2198 to 2226) (see Fig. S5 in the supplemental material). The sequence of a potential rho-independent terminator starts 42 bp downstream of chi (positions 2070 to 2096) (see Fig. S5).

Expression of chi in E. coli and characterization of the recombinant enzyme with respect to its flavanonol-isomerizing activity.

The gene chi, including the proposed promoter and terminator sequences was unidirectionally cloned using a lac promoter-containing vector for its heterologous expression in E. coli. The expression of chi was tested with and without induction by IPTG in selected clones carrying the encoding gene in either direction. In the clone carrying chi codirectional with the lac promoter (E. coli pTI2), gene expression was already observed in the absence of IPTG and was increased up to 2.4-fold in its presence. Maximal taxifolin-converting activity of 169 ± 1 μmol min⁻¹ (mg protein)⁻¹ was reached in the cell extract prepared from cultures harvested 4 h after induction by IPTG. The clone carrying the gene in the opposite direction to the lac promoter (E. coli pTI3) also expressed chi, which indicates the functionality of the native on-board promoter upstream of chi. Maximal taxifolin-converting activity of 55 ± 1 μmol min⁻¹ (mg protein)⁻¹ was observed in the resulting cell extract after 20 h of growth. As expected for this clone, gene expression was not further induced by adding IPTG. Compared to E. coli pTI3 and the clone carrying the empty vector, E. coli pTI2 showed an initial delay when grown in liquid medium and smaller colonies on agar plates, in particular in the presence of IPTG.

The recombinant CHI in the cell extract of highly expressing E. coli pTI2 (see Fig. S6, lane 2, in the supplemental material) was analyzed with respect to its apparent molecular masses in SDS-PAGE (31 kDa), native PAGE (130 kDa), and gel filtration (270 kDa) as well as its temperature optimum (41°C) and pH optimum (5.2 to 8.0). The K_m for (+)-taxifolin was 4.1 ± 0.6 μM. (+)-Dihydrokaempferol and the chalcone butein were also converted by recombinant CHI, but at lower activities of 60 and <1%, respectively, compared to (+)-taxifolin. (−)-Taxifolin and (−)-dihydrokaempferol did not serve as the substrates but reduced the conversion of the corresponding (+)-enantiomer at equimolar concentrations to 56 and 45%, respectively. Overall, the characteristics of the recombinant CHI were very close to those of the native enzyme purified from E. ramulus.

As for the native enzyme, transformation of naringenin or eriodictyol by recombinant CHI was not observed. However, these flavanones inhibited the CHI-catalyzed isomerization of the corresponding flavanonols. Thus, the rate of (+)-taxifolin conversion was reduced to 51% by adding eriodictyol at an identical concentration, and the (+)-dihydrokaempferol conversion rate was reduced to 84% in the presence of naringenin (Fig. 3).

FIG 3.

Inhibition of CHI-catalyzed flavanonol conversion by structurally analogous flavanones. The time course of the conversion of flavanonols (180 μM) by cell extracts of chi-expressing E. coli pTI2 in the absence or presence of the corresponding flavanones (180 μM) is shown. (A) Conversion of (+)-taxifolin (●) to alphitonin (▲) in the absence of eriodictyol and conversion of (+)-taxifolin (○) to alphitonin (△) in the presence of eriodictyol. (B) Conversion of (+)-dihydrokaempferol (●) to maesopsin (▲) in the absence of naringenin and conversion of (+)-dihydrokaempferol (○) to maesopsin (△) in the presence of naringenin.

Molecular docking of (+)- and (−)-taxifolin into the substrate-binding site of CHI.

For the docking studies, the published X-ray crystal structure of the ligand-free CHI at a 2.8-Å resolution (PDB ID no. 3ZPH) was used (15). A hydrogen bond network involving Asp79 and Gln101 was suggested to be crucial for the suitable orientation of the substrate naringenin within the active site (15). Accordingly, we took a molecular docking approach with (+)-taxifolin and (−)-taxifolin, respectively. To generate putative complexes of CHI with the two taxifolin enantiomers, the active-site area was specified by placing a grid box comprising Asp79 and Gln101 as well as the approximate binding pockets. This procedure yielded reasonable orientations for both ligands (+)-taxifolin and (−)-taxifolin (Fig. 4). In the predicted (+)-taxifolin complex (Fig. 4A), the two phenolic groups of the B-ring form a bifurcated hydrogen bond to Asp79. The 5-hydroxy and 7-hydroxy groups of the A-ring interact via two hydrogen bonds with the carboxamide residue of Gln101. Moreover, the carbonyl oxygen of (+)-taxifolin acts as a hydrogen bond acceptor for the hydroxy group of Thr71. This assumed orientation holds the substrate in the binding pocket, where the catalytic His33 residue is located in its central upper part. Essentially the same interactions are proposed for the occupation of (−)-taxifolin in the substrate-binding site of CHI (Fig. 4B), involving the side chains of Asp79, Gln101, and Thr71 for a hydrogen bond network. Docking of (+)-taxifolin and (−)-taxifolin into the ligand-bound structure of CHI (PDB ID no. 4D06) (17) revealed exactly the same binding mode predicted for both enantiomers in the ligand-free CHI.

FIG 4.

Docking of taxifolin enantiomers into the substrate-binding site of CHI. Putative binding mode of (A) (+)-(2R,3R)-taxifolin (colored cyan) and (B) (−)-(2S,3S)-taxifolin (colored orange) in the substrate-binding site. The active site is shown in the gray surface representation. Oxygen atoms are labeled in red, nitrogen atoms in blue, and selected hydrogen atoms in white. The arrows indicate the distance between the catalytic histidine residue and the hydrogen atom at the 3 position of the respective taxifolin enantiomer.

Although the accommodations of (+)-taxifolin and (−)-taxifolin in the substrate-binding site of CHI are similar (see Fig. S7 in the supplemental material), the accessibility of the hydrogen at the 3 position for His33 differs considerably. In case of (+)-taxifolin, this hydrogen points toward the upper part of the binding site and has a short distance of 2.17 to 2.39 Å to the N^ε2 atom of His33, depending on the rotation of the imidazole ring. Since in (−)-taxifolin, the configurations at positions 2 and 3 are inverted, the position 3 hydrogen is oriented to the lower part of the binding pocket, away from His33. Based on these docking results, we assumed that His33 also functions as crucial residue for the flavanonol-isomerizing activity of CHI. This suggests a general acid-base catalysis as reported for the flavanone-converting activity of CHI (17). A transfer of the proton at position 3 of (+)-taxifolin to His33 would facilitate the subsequent transformation to alphitonin, in accordance with the observed stereospecificity of taxifolin isomerization.

Mutagenesis of the catalytic histidine in CHI.

Based on the above-described docking results, the histidine at position 33 of CHI (position 34 referring to the amino acid sequence before posttranslational cleavage of the N-terminal methionine) (14, 17) was replaced by alanine using site-directed mutagenesis. The mutated chi was heterologously expressed in E. coli at a similar level to the wild-type enzyme (see Fig. S6, lane 3, in the supplemental material). The migration behavior of the mutant CHI in native PAGE analysis was in agreement with that of wild-type CHI, which confirmed its stability. The H33A modification led to a complete loss of flavanonol isomerization activity as tested with (+)-taxifolin and (+)-dihydrokaempferol. Even at a protein concentration of 0.2 mg ml⁻¹, which is increased 1,000-fold compared to the concentration used in assays with the wild-type CHI, no formation of alphitonin from taxifolin by the mutant CHI was observed (Fig. 5).

FIG 5.

Complete activity loss of the mutant CHI. Incubation of (+)-taxifolin (●) with cell extract of chi-expressing E. coli pTI2 (final protein concentration, 0.2 μg ml⁻¹) led to the formation of alphitonin (▲). No conversion of (+)-taxifolin (○) was observed with cell extract of E. coli pTI2-MUT expressing the mutated chi, even at a 1,000-fold-increased protein concentration (200 μg ml⁻¹).

Functional expression of the flavanonol-converting enzyme from Flavonifractor plautii in E. coli.

A hypothetical protein from the completely sequenced, flavonoid-degrading F. plautii showed 50% sequence identity to the CHI from E. ramulus (see Fig. S4 in the supplemental material), suggesting a similar function in this bacterium. Its amino acid sequence predicted a cytosolic localization of the protein. The encoding gene was cloned and expressed in E. coli. The resulting soluble protein was subsequently purified from the cell extract at a yield of 38% by means of the fused C-terminal Strep tag. The protein did indeed convert (+)-taxifolin to alphitonin, but with a low specific activity of 8.50 ± 1.15 nmol min⁻¹ (mg protein)⁻¹ only. The recombinant F. plautii enzyme also isomerized (+)-dihydrokaempferol to maesopsin. This reaction proceeded at 126% of the rate observed for conversion of (+)-taxifolin. Expression of the enzyme with an N-terminal Strep tag was less effective and resulted in a yield of only 3% of purified protein. This N-terminally tagged enzyme showed a taxifolin-converting activity of 12.2 ± 0.6 nmol min⁻¹ (mg protein)⁻¹.

DISCUSSION

Taxifolin is present in onions and pharmacologically active plant extracts and is considered to have chemopreventive effects against cancer, cardiovascular disease, and liver disease (26). The naturally occurring (+)-enantiomer has a (2R,3R)- configuration (24, 27). Moreover, taxifolin represents the first intermediate in the conversion of quercetin by gut bacteria (11, 12). E. ramulus has been identified as a prevalent human gut bacterium involved in the conversion of dietary flavonoids (10, 28–30). In the course of elucidating the pathway of quercetin conversion by E. ramulus, alphitonin was discovered as another intermediate beside taxifolin, and an isomerase activity converting taxifolin to alphitonin was subsequently recovered in cell-free preparations (11). Here, we describe the purification of the responsible enzyme. Several characteristics of the taxifolin-isomerizing enzyme, such as molecular mass, chalcone-cyclizing activity, and the N-terminal sequence, matched those of the CHI from E. ramulus. This enzyme was characterized previously as the first bacterial counterpart (14) of the well-known chalcone isomerases from plants (31–33). Based on the sequences of the taxifolin-isomerizing enzyme and CHI from E. ramulus DSM 16296, the latter sequence published recently (15), the identities of the two enzymes were finally confirmed.

The genome of the E. ramulus type strain (ATCC 29099) also contains a gene that, based on 99% identity of its product to the enzyme from E. ramulus DSM 16296, encodes a putative CHI. The CHI sequence displays much lower similarity to putative bacterial acetyl-CoA hydrolases, the functional assignment of which has not yet been verified. The hypothetical protein from F. plautii with 50% identity to CHI was hypothesized to represent also a flavanonol/flavanone isomerase, since F. plautii is known to metabolize flavonols and flavones via the same pathways described for E. ramulus (12). Heterologous expression of the F. plautii gene product confirmed its ability to convert flavanonols, although at much lower rates than CHI from E. ramulus. The reduced activity may be explained by impaired gene expression in E. coli and/or the presence of the tag, which was attached to facilitate subsequent enzyme purification. Fused to the C terminus, the tag probably disturbed dimerization, which could be based on the seven conserved C-terminal residues as shown for the CHI from E. ramulus (17). Being in relatively close proximity to the catalytic domain, the N-terminal tag may have a negative impact on catalysis.

Recently, Thomsen et al. identified the active site of the CHI from E. ramulus by cocrystallization with its flavanone substrate (S)-naringenin (17). The structure of this complex revealed the central role of His33 in the general acid-base catalysis, which was supported by mutagenesis (17). The identity of CHI investigated by Herles et al., Gall et al., and Thomsen et al. (14, 15, 17) with the taxifolin-isomerizing enzyme enabled us to provide a comprehensive picture of the remarkable reactions catalyzed by this enzyme. The predicted mechanism of the ring contraction of flavanonols, compounds 1, to auronols, compounds 6, catalyzed by CHI is depicted in Fig. 6. The essential role of the His33 residue was confirmed by demonstrating the complete loss of the flavanonol-isomerizing activity of the H33A mutant. The conversion is initiated by a proton transfer from substrate 1 to the active-site histidine. As concluded from the docking results, the deprotonation occurs at the C-3 carbon and does not affect the 3-hydroxy group, which is expected to be similarly acidic but whose deprotonation would not initiate the observed transformation of the substrate. The generated negative charge of the carbanion, compound 2, facilitates ring opening to a hydroxychalcone, compound 3, which possibly undergoes protonation at the phenolate oxygen to form a neutral chalcone intermediate, compound 4. So far, the mechanism is essentially the same as elucidated by Thomsen et al. for the conversion of (S)-naringenin to naringenin chalcone (17). Noteworthy, it was demonstrated by NMR H/D exchange experiments that the (S)-naringenin proton H_3b at the pro-S position (structure in Fig. 1) is involved in the catalytic cycle of naringenin-to-naringenin chalcone conversion (17). In flavanonol transformation, the corresponding proton (i.e., the proton in the cis position relative to the 2-aryl residue) is transferred to the active-site histidine. Accordingly, (+)-taxifolin and (+)-dihydrokaempferol were identified as the substrates for CHI, but (−)-taxifolin and (−)-dihydrokaempferol with the 3-proton in the trans position to 2-aryl were not converted. Thus, the proposed mechanism of the flavanonol conversion by CHI is strongly supported by the pronounced stereospecificity of the enzyme, which has been observed in the course of our investigation.

FIG 6.

Proposed mechanism of flavanonol conversion by CHI. The acid-base catalysis is mediated by the active-site histidine residue. Compound 1, R = OH for (+)-taxifolin and R = H for (+)-dihydrokaempferol; compound 6, R = OH for alphitonin and R = H for maesopsin.

The further transformation of the ring-opened intermediate depends on the substitution at C-3 of the flavonoid substrate. In flavanones, such as naringenin, the corresponding chalcone lacks the α-hydroxy group, which is present in compound 4. Hence, the flavanone-derived chalcone, such as naringenin chalcone (structure in Fig. 1), represents the product of the isomerase-catalyzed reaction (14, 15). However, those chalcones are susceptible to recyclization, an intramolecular oxa-Michael addition which proceeds as a 6-endo-trig cyclization. For this reason, the characterization of the enzymatic activity was performed by analyzing the backward reaction of chalcones to flavanones (14) or by coupling the CHI with an ERED (15). A different fate results for the intermediate α-hydroxychalcones, compounds 4, derived from flavanonols. Tautomeric rearrangement to 1,2-diketones, compounds 5, and subsequent cyclization to a five-membered ring produce the final auronol-type compounds 6, e.g., alphitonin and maesopsin. This unique ring contraction through a 5-exo-trig cyclization is driven by the formation of the thermodynamically favored product 6, while the backward reaction via a 6-endo-trig cyclization to compound 1 is negligible. The catalytic efficacy of CHI becomes apparent when considering the drastic conditions that are required for conversion of a flavanonol, such as taxifolin, to the corresponding auronol, alphitonin, by a nonenzymatic reaction (19, 27). For the thermal taxifolin-alphitonin rearrangement at 115°C, monitored over a period of 2 weeks, a half-life for taxifolin of 54 h was determined (19).

The enzyme-catalyzed transformation of either flavanones or flavanonols leads to different types of products. The former substrates possess a methylene group as part of the C-ring, while one methylene hydrogen is replaced by OH in the latter. It is this critical difference in the substrates' molecular structures that defines the reaction course. In case of flavanonol conversion (Fig. 6), the inherent possibility of generating a 1,2-diketone intermediate, compound 5, which serves as an electrophilic trap, enables the ring contraction to compound 6. However, CHI is not considered to exhibit pronounced substrate promiscuity. The terminal phenol groups at rings A and B are required for the formation of an enzyme-substrate complex and subsequent transformation. This is supported by our finding that flavanonol (structure in Fig. 1) was not converted by CHI. The reduced activity observed for (+)-dihydrokaempferol compared to (+)-taxifolin may be explained by diminished substrate recognition due to the lack of the second hydroxy group in the B-ring. The crucial stereochemistry of the substrates becomes obvious when investigating the two enantiomers of taxifolin and dihydrokaempferol. These four compounds are all expected to be accommodated in the active site of CHI. However, we demonstrated that only binding of the (+)-enantiomers is productive. If so, the corresponding (−)-enantiomers should compete with the substrates for the binding site and act as enzyme inhibitors. This assumption was confirmed by demonstrating that the alphitonin formation is inhibited by (−)-taxifolin and the maesopsin formation by (−)-dihydrokaempferol. Moreover, eriodictyol inhibited the generation of alphitonin and naringenin that of maesopsin. Thus, the corresponding flavanones compete as alternate substrates with their flavanonol counterparts for binding to the active site of CHI.

Our studies subsequently revealed that a single isomerase plays a dual role in bacterial flavonoid conversion. It catalyzes a key step in the degradation of both flavonols (via flavanonols) and flavones (via flavanones). Bifunctionality is widespread among bacterial enzymes due to their compact genomes (34–38). However, CHI is not a classical bifunctional enzyme, as it does not utilize two different domains for catalysis. The degradation pathway of the two flavonoid classes branches at this point due to the formation of either an auronol or a chalcone product. Strictly speaking, after essentially consistent binding and ring cleavage of the particular type of substrate, the fate of the ring-opened intermediate is driven by the opportunity to either generate the thermodynamically favored five-membered ring or not, thus directing the subsequent steps of further conversion into two different pathways. Hence, CHI can be considered to exhibit a certain catalytic promiscuity (39), where the reaction path is determined by the substrate structure. While the conversion of auronols has not been studied in detail, the chalcones may be further transformed in E. ramulus by ERED and Phy (15, 16). According to our studies and the completed sequence of the E. ramulus type strain, the genes encoding the involved enzymes are not clustered but distributed in the genome. This makes it even more challenging to identify the remaining enzymes, which, however, is essential to elucidate the complete conversion of these bioactive flavonoids by intestinal bacteria in the human gut.