Discovery of a sesamin-metabolizing microorganism and a new enzyme

Takuto Kumano; Etsuko Fujiki; Yoshiteru Hashimoto; Michihiko Kobayashi

doi:10.1073/pnas.1605050113

New Research In

Edited by Julian Davies, University of British Columbia, Vancouver, BC, Canada, and approved June 14, 2016 (received for review March 28, 2016)

Significance

Lignans, including sesamin, are produced by a wide variety of plants, but the microbial degradation of lignan has not been identified biochemically. Here, we show that Sinomonas sp. no. 22 can catabolize sesamin as a sole-carbon source. We identified the sesamin-converting enzyme, SesA, from strain Sinomonas sp. no. 22. SesA catalyzed methylene group transfer from sesamin to tetrahydrofolate (THF). The resulting 5,10-CH₂-THF might find use as a C1-donor for bioprocesses. SesA gene homologs were found in the genomes of both Gram-positive and Gram-negative bacteria, suggesting that sesamin (lignan) utilization is a widespread, but still unrecognized, function in environments where lignans are produced and degraded.

Abstract

Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: K_m for sesamin = 0.032 ± 0.005 mM, V_max = 9.3 ± 0.4 (μmol⋅min⁻¹⋅mg⁻¹), and k_cat = 7.9 ± 0.3 s⁻¹. Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (−)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.

We have been involved in studies of not only microbial metabolism of man-made compounds (1 ⇓–3) but also biologically active natural compounds, such as curcumin (4). In this study, we characterize the microbial metabolism of the lignan sesamin.

Lignans (5) are plant-derived compounds consisting of dimers of phenylpropane units (6). They are found in a wide variety of plant-based foods. Whole-grain products, vegetables, fruits, nuts, seeds, and beverages such as tea, coffee, and wine are dietary sources of lignans. In Asian countries, sesame, which contains lignans, is used traditionally as a food. A major lignan is sesamin, which is a biologically active compound with antioxidative (7), cholesterol-lowering (8), lipid-lowering (9), antihypertensive (10), and antiinflammatory (11) properties.

In humans, sesamin is metabolized by CYP450 enzymes into sesamin mono- and di-catechol in liver microsomes (12). Sesamin monocatechol is metabolized further by UDP-glucuronosyltransferase (UGT) and O-methyl transferase (COMT) (13), and the resulting glucuronides of sesamin metabolites are excreted in the bile and urine (14).

In microorganisms, on the other hand, metabolism of sesamin has been reported in few species. Aspergillus oryzae converts sesamin to sesamin mono- and di-catechol (15) whereas intestinal bacteria convert sesamin to the so-called “mammalian lignans” enterodiol and enterolactone (16). The activities of these metabolites make them useful dietary substances. Sesamin mono- and di-catechol show stronger antioxidant activity than sesamin (17). Also, mammalian lignans show other properties: higher antioxidant activity than vitamin E (18); reduction of cardiovascular disease risk (19); reduction of the risk of arteriosclerosis by decreasing the total cholesterol level (20); and reduction of inflammation markers (21). However, neither sesamin-metabolizing enzymes nor their genes have been identified in microorganisms, including intestinal bacteria and A. oryzae.

Here, we describe the isolation and identification of a sesamin-catabolizing soil microorganism, Sinomonas sp. no. 22, together with purification and characterization of a previously unknown sesamin-modifying enzyme. This enzyme has the unique catalytic ability to transfer the methylene group from sesamin to tetrahydrofolate (THF) through ring cleavage (Fig. 1). In addition, we clarify the biochemical properties of this enzyme and propose a possible reaction mechanism. Our findings provide novel insights into the catabolism of natural compounds and THF-dependent metabolic pathways.

Fig. 1.

Schematic representation of the bond cleavage location in the structure of substrates for SesA (A) and previously reported THF-dependent methyl transferases (B). The cleavage site of the bond between atoms within a substrate is indicated by a dashed line.

Results

Isolation and Identification of Sesamin-Metabolizing Bacteria.

The soil samples used for microbial screening were collected from sesame gardens at the University of Tsukuba. At ∼1 mo from the start of this study, by using the enrichment culture method described in Materials and Methods, we isolated 40 microorganisms that were able to grow on culture medium containing sesamin as the sole carbon source. We selected one isolate, strain no. 22, for further study. This strain showed 99% 16S rRNA gene sequence similarity to Sinomonas atrocyanea DSM20127^T. Morphological and biochemical properties of strain no. 22 are shown in SI Appendix, Supplementary Data.

Structure Determination of the Sesamin Metabolites.

We incubated a cell-free extract of strain no. 22 with sesamin as a substrate. Two products (compounds A and B) were isolated by ethyl acetate extraction and preparative HPLC (Fig. 2).

Fig. 2.

HPLC analysis of reaction products of sesamin. Chromatogram of the reaction mixture after incubation of sesamin with a cell-free extract of strain no. 22. The products are indicated as A and B.

High-resolution mass spectrometry (HRMS) analysis in the negative mode revealed the molecular ion of compound A at m/z 341.1029 [M-H]⁻, which was in agreement with the calculated mass of C₁₉H₁₇O₆, and that of compound B at m/z 329.1022 [M-H]⁻, in agreement with the calculated mass of C₁₈H₁₇O₆ (SI Appendix, Fig. S1). The loss of 12 atomic mass units suggested modification of the methylenedioxyphenyl group.

¹H NMR spectra of compounds A and B showed proton signals corresponding to the tetrahydrofuran ring and 1,3,4-substituted benzene moiety expressed (SI Appendix, Fig. S1). On the other hand, the proton signal corresponding to H-10′ of sesamin was absent in compound A (SI Appendix, Figs. S1 and S2). The proton signals corresponding to H-10 and H-10′ of sesamin were not observed for compound B (SI Appendix, Figs. S1 and S3).

Based on these observations, compounds A and B were identified as sesamin monocatechol (1 in Fig. 3) and sesamin di-catechol (2 in Fig. 3), respectively (22).

Fig. 3.

SesA activities for plant-derived methylenedioxyphenyl compounds and their derivatives. The specific activities for each substance and structures of reaction products are indicated.

Purification of the Sesamin-Metabolizing Enzyme.

The sesamin-metabolizing enzyme was purified 1.2-fold with a yield of 3.4% (SI Appendix, Table S3). Considering the relationship between the protein amount and the activity of each fraction obtained on gel-filtration chromatography, the target enzyme was as the ∼50-kDa protein band observed on SDS/PAGE (SI Appendix, Fig. S4 and Table S4). An N-terminal amino acid sequence of this protein was determined to be TAEQAIN.

The gene for the sesamin-metabolizing enzyme, named SesA, was identified from the draft genome sequence data for strain no. 22 (see Identification of the Sesamin-Metabolizing Enzyme). The primary structure of SesA suggested that it might require THF as a cofactor. On the addition of THF to cell-free extracts, the sesamin-metabolizing activity increased 350 times (Table 1 and SI Appendix, Table S3).

View this table:

Table 1.

Purification of SesA

SesA was purified as a single band on SDS/PAGE (Table 1 and SI Appendix, Fig. S5) after ammonium sulfate precipitation (35–55%) and column chromatography (TOYOPEARL Butyl 650M, Mimetic Orange 1 A6XL). The molecular mass of native SesA was 150 kDa, (SI Appendix, Fig. S6). The molecular mass of SesA was calculated to be 50,385 Da, which was consistent with that of the purified enzyme determined by SDS/PAGE. These results indicate that SesA consists of three identical subunits.

Identification of the Sesamin-Metabolizing Enzyme.

We determined the draft genome sequence for strain no. 22 using a next-generation sequencer, Hiseq2500 (Illumina). From the sequence information, we identified an ORF of 1,359 nucleotides, 21 of which corresponded to the above N-terminal sequence (SI Appendix, Supplementary Data 2). The deduced amino acid sequence identified a putative folate-binding domain as in the glycine cleavage T-protein (GcvT) (pfam01571).

Time-Dependent Sesamin Metabolism During Cultivation.

The growth curve of strain no. 22 is shown in SI Appendix, Fig. S7. In liquid medium containing sesamin as a sole-carbon source (SI Appendix, Fig. S7A), the specific activity of the enzyme increased after 8 h cultivation, followed by a decrease in sesamin in the medium and an increase in the protein compared with cell growth. On the contrary, in medium containing glucose as sole-carbon source, cells grew exponentially for 4 h, but sesamin-converting activity was not observed during cultivation. Furthermore, in 2× YT liquid medium, sesamin-converting activity was detected only in the presence of added sesamin (SI Appendix, Fig. S7B).

Western Blot Analysis.

To confirm correlation of SesA expression with sesamin metabolism, the induction of SesA in the presence of sesamin was followed by Western blot analysis on cell-free extracts in the following culture conditions: One was in 0.1% (wt/vol) sesamin-supplemented 2× YT liquid medium, and the other was in in 2× YT liquid medium. A cross-reacting band was observed only in the cell-free extract of cells cultured with sesamin (SI Appendix, Fig. S7D). In addition, SesA enzyme activity (0.053 units/mg) was observed only in cells grown in the presence of sesamin.

Cloning and Heterologous Expression of sesA and Biochemical Properties of SesA.

The 1.4-kb region of the SesA-coding gene was inserted into an expression vector, and recombinant SesA was produced in Escherichia coli and purified. The specific activity of the recombinant SesA was approximately the same (8.8 units/mg) as that of SesA purified from strain no. 22.

We examined the effects of temperature and pH on SesA activity. The optimal reaction temperature and pH were below 40 °C and pH 7.5–8.5, respectively (SI Appendix, Fig. S8 A and B). SesA was most stable under 30 °C and in the pH range of 5.5–10.0 (SI Appendix, Fig. S8 C and D).

The absorption spectrum of the purified SesA showed an absorbance maximum near 280 nm. No other absorption peak or shoulder was observed at higher wavelengths (SI Appendix, Fig. S9). These results suggest that no cofactor is bound to the purified enzyme. The CD spectrum is shown in SI Appendix, Fig. S10. Qualitative analysis of metal content was performed by inductively coupled plasma atomic emission spectroscopy analysis. The enzyme contained 1.70 mol of phosphorus and 0.70 mol of sulfur per mole of subunit. No other metal was detected within the assay limits (SI Appendix, Supplementary Data 3).

Stoichiometry.

The stoichiometry of the SesA reaction was examined. The amounts of sesamin, sesamin monocatechol, sesamin di-catechol, and folates were determined by HPLC and liquid chromatography (LC)/MS/MS. After 30 min incubation, sesamin mono- and di-catechol increased to 77 and 9.3 μM, respectively. Coincidentally, sesamin decreased by 80 μM (SI Appendix, Fig. S11A).

We detected 5,10-CH₂-THF in SesA reaction mixtures. After 30-min incubation, 5,10-CH₂-THF increased to 85 μM (SI Appendix, Fig. S11B); there was no change in the 5-CH₃-THF amount. THF could not be determined because of its instability under our assay conditions. These results demonstrate that sesamin mono-catechol, di-catechol, and 5,10-CH₂-THF were formed stoichiometrically with the consumption of sesamin during the enzymic reaction (Fig. 4A).

Fig. 4.

Sesamin metabolic pathway and proposed reaction mechanism. (A) Proposed sesamin metabolic pathway in strain no. 22. (B) DNA sequence of region encoding SesA and its flanking region. The thfl gene exhibits 60% amino acid sequence identity with that of Mycobacterium bovis (UniProtKB accession code P0A5T7), and the thf2 gene exhibits 69% amino acid sequence identity with that of Clavibacter michiganensis (UniProtKB accession code B0RD2). (C) Proposed reaction mechanism for SesA. In C, “B” represents a base.

Kinetic Analysis.

Using varying concentrations of sesamin in the presence of 1 mM THF, a typical hyperbolic curve of product formation over substrate concentration was obtained (SI Appendix, Fig. S11C), indicating that the reaction followed Michaelis–Menten kinetics. Apparent steady-state kinetic constants were estimated for sesamin by quantitative measurements of sesamin monocatechol by HPLC analysis. Nonlinear regression analysis revealed the following: K_m = 0.032 ± 0.005 mM, V_max = 9.3 ± 0.4 (μmol⋅min⁻¹⋅mg⁻¹), and k_cat = 7.9 ± 0.3 s⁻¹.

Substrate Specificity.

To examine the substrate specificity of SesA, we investigated the compounds listed in Fig. 3. The structures of the products were determined by LC/MS/MS and NMR analyses. SesA catalyzed the demethylenation of (+)-episesamin, (−)-asarinin, sesaminol, and (+)-sesamolin to yield monocatechols 3 (SI Appendix, Figs. S12–S16), 4 (SI Appendix, Figs. S17–S22), 6 (SI Appendix, Figs. S23–S25), and 8 and 9 (SI Appendix, Figs. S26–S28), and di-catechols 5 (SI Appendix, Figs. S17 and S29–S32), 7 (SI Appendix, Fig. S23), and 10 (SI Appendix, Fig. S26). Moreover, piperine (derived from black pepper) was also demethylated (SI Appendix, Figs. S33–S35). SesA activity was specific for the methylenedioxyphenyl and not the methoxyphenyl group.

Mutational Analysis of SesA.

To investigate the reaction mechanism of SesA, we constructed a set of mutants with single-amino acid substitutions. SesA was found to belong to a diverse family of enzymes that include specific domains of dimethylglycine oxidase (DMGO) (23), sarcosine oxidase (24), dimethylsulfoniopropionate demethylase (DmdA) (25), and demethylase. The enzyme acts on lignin-degradation products such as syringate and vanillin [DesA (26, 27) and LigM (28), respectively], as well as GcvT (29).

Based on the amino acid sequence alignment of these enzymes, we prepared three mutants of SesA: D95A, E189A, and Y221A. Each of the mutant enzymes was expressed in E. coli and purified by the method described in Materials and Methods (SI Appendix, Fig. S36). Enzymatic activity was measured using the method as that for wild-type SesA. E189A and Y221A exhibited no activity at all. The activity of the D95A derivative was 40% compared with that of the wild-type enzyme.

Discussion

In nature, many physiologically active compounds, such as flavonoids, terpenoids, alkaloids, steroids, coumarins, glycosides, and nucleosides, are produced by plants and microorganisms. Unique pathways of microbial metabolism of these compounds have been reported (30 ⇓⇓⇓–34). Research on the microbial metabolism of a diversity of natural compounds can be expected to reveal novel enzymes (4) and catalytic functions (34). We recently reported a curcumin metabolic pathway in E. coli and identified a novel curcumin/dihydrocurcumin reductase (4). Here, we isolated sesamin-catabolizing microorganisms and determined the initial steps of the sesamin-metabolic pathway at both protein and gene levels.

Sesamin is a major lignan in sesame oil with characteristic methylenedioxyphenyl groups. A methylenedioxyphenyl group is present in some plant metabolites, such as berberin (isolated from Berberis), piperonal (isolated from dill, vanilla, violet flowers, and black pepper), and piperine (isolated from black pepper), as well as sesamin. Also, drugs such as tadalafil and 3,4-methylenedioxymethamphetamine (MDMA) have a methylenedioxyphenyl group. In humans, the methylenedioxy bridges (O-C-O) of methylenedioxyphenyl compounds are oxidized to catechols by CYP450s (12, 35).

On the other hand, microbial enzymes that metabolize methylenedioxyphenyl bridges have not been reported although metabolites have been identified in a few cases. For example, sesamin is converted into sesamin monocatechol and sesamin di-catechol by A. oryzae (15). In addition, cell-free extracts of Pseudomonas fluorescens strain PM3 oxidize piperonylic acid into protocatechuate and formic acid, requiring NADH or NADPH as a cofactor (36). Recently, it was reported that intestinal bacteria convert sesamin into mammalian lignans (16).

In this study, we identified SesA, which converts sesamin into sesamin mono- and di-catechol. The cleaved methylene group of sesamin is transferred to THF, 5,10-CH₂-THF being formed. Thus, SesA is a THF-dependent sesamin/sesamin-monocatechol methylenetransferase. In this reaction, the methylene group from the methylenedioxy bridge is transferred without an apparent change in redox state to THF. This mechanism is distinct from that of CYP450 (12), which oxidatively removes the methylene group of sesamin. Compared with the enzymatic activity of SesA and CYP2C9, which contributes significantly to the metabolism of sesamin in the liver, the K_m values of SesA and CYP2C9 were 32 μM and 5.4 μM, respectively. On the other hand, the k_cat value of SesA for sesamin was 220 times higher than that of CYP2C9 (12).

Homology searches of the protein database demonstrated that SesA exhibits low similarity (∼20%) to GcvT, which is involved in the glycine cleavage system (GCS) together with P-protein, H-protein, and L-protein. In general, GCS is found in most bacteria, archaea, and the mitochondria of all eukaryotes and plays critical roles in both glycine degradation and one-carbon metabolism (37).

The folate-binding domain of GcvT (pfam01571) is conserved in SesA and also in DMGO, sarcosine oxidase, DmdA, DesA, and LigM. These GcvT family proteins exhibit weak sequence identities and are classified into separate clades from one another (SI Appendix, Fig. S37).

SesA is distinct as follows: (i) SesA is a homo-trimer that forms no complex with any other proteins; (ii) the substrates of SesA are aromatic compounds; and (iii) SesA catalyzes a ring cleavage reaction to transfer the methylene group to THF (Fig. 1). In particular, point iii is unique to SesA among the GcvT family proteins.

SesA, LigM, and DesA all metabolize aromatic compounds although the sequence similarities of SesA to LigM and DesA are only 22% and 26%, respectively. On the other hand, SesA showed distinct enzymatic activities compared with LigM and DesA. LigM and DesA transfer the “methyl” group to THF, yielding “5-CH₃-THF.” On the contrary, SesA transferred the “methylene” group of sesamin to THF, giving “5,10-CH₂-THF.” Although we investigated each of the compounds listed in Fig. 3, we found that SesA, does not transfer the methyl group to THF.

The enzymatic activity of SesA was influenced by the stereochemistry of the methylenedioxyphenyl group (Fig. 3). Diasesamin and (+)-episesamin monocatechol are inert substrates (Fig. 3), suggesting that SesA catalyzes only the demethylenation of an equatorial methylenedioxyphenyl group, when the stereochemistry of the tetrahydrofuran ring is 8R, 8′R. On the other hand, (−)-asarinin is converted into (−)-asarinin di-catechol, which suggests that SesA is able to catalyze the demethylenation of methylenedioxyphenyl groups that are in both axial and equatorial positions, when the stereochemistry of the tetrahydrofuran ring is 8S, 8′S.

Also, the activity of SesA was affected by substrate size. Piperine was an active substrate whereas small methylene dioxyphenyl compounds such as samin were inert substrates (Fig. 3).

The crystal structure of GcvT of Thermotoga maritima shows that aspartic acid (D96), glutamic acid (E195), and tyrosine (Y100) are hydrogen-bonded to THF (29). These hydrogen bonds are also observed in the structures of DMGO, sarcosine oxidase, and DmdA; sequence alignment of SesA, LigM, DesA, GcvT, DmdA, and DMGO demonstrated that D95 and E189 of SesA corresponded to D96 and E195 of GcvT (SI Appendix, Fig. S38). Tyrosine is not conserved at the corresponding position in the protein sequences of GcvT family proteins. Strictly speaking, Y100 of GcvT, Y660 of DMGO, and Y206 of DmdA are hydrogen-bonded to THF. In this alignment, Y221 of SesA corresponded to Y247 and Y242 of LigM and DesA, respectively. The E189A and Y221A mutants of SesA were inactive. Considering the reported crystal structures of DmdA (25) and GcvT (29), this finding suggests that E189 and Y221 of SesA would be hydrogen-bonded to THF.

The reaction mechanisms of GcvT, DmdA, and DMGO were proposed based on 3D structural and mutational analyses (23, 25, 38). (i) In GcvT of E. coli, the electron relay from D97 (or D96) to N113 through the hydrogen bond could make N113 act as a base to deprotonate the protonated amino group of the aminomethyllipoyllysine arm in the substrate, followed by the cleavage of the C–S bond of the arm after migration of a proton from the protonated R223 to the substrate, yielding a reactive iminium intermediate (38). The iminium intermediate reacts with the N5 atom of THF to form ammonia and an iminium ion including the N5 atom of THF. Next, D97 deprotonates the N10 atom of 5-CH₂-THF, and this deprotonated N10 attacks the iminium ion including N5, to form 5,10-CH₂-THF. (ii) In DmdA, methyl transfer is suggested to be coupled with proton transfer that is initiated by a base and mediated by a water molecule in the active site. In this reaction, as a nucleophile, Sp³ hybridized-N5 of THF attacks the CH₃ group on the sulfonium ion of the substrate, to yield 3-(methylthio)propionic acid (25). Therefore, the proton donor is not required in this reaction. (iii) In DGMO, THF attacks the iminium ion of the substrate via the nucleophilic N10 atom, with concomitant deprotonation by D552, followed by the formation of sarcosine and 5,10- CH₂-THF through intramolecular rearrangement of the covalent intermediate formed between THF and the iminium intermediate (i.e., N5 of THF attacks on the covalent intermediate with concomitant deprotonation of N5 of THF by the nascent sarcosine) (23).

In our study, on the other hand, the activity of D95A was found to be 40% less compared with that of the wild-type enzyme. Considering this finding and the proposed reaction mechanisms of other GcvT family enzymes, direct nucleophilic attack on sesamin by N5 of THF would initiate the reaction, as seen in the case of DmdA. However, the reaction mechanism of SesA is not the same as other members of the GcvT family. In the SesA reaction, the methylenedioxy groups (O–C–O) are cleaved to yield OH groups of catechol moieties; proton donation is required to cleave the O–C–O bond, which is not present in substrates of other GcvT family enzymes. Therefore, we propose a possible reaction mechanism, in which proton donors (indicated by BH⁺ and B′H⁺ in Fig. 4C) are involved. In previously reported reaction mechanisms of GcvT family enzymes, proton donation does not occur except in GcvT. In GcvT, R223 is predicted to donate a proton to the substrate for cleavage of the C–S bond. According to the amino acid alignments of these proteins, an arginine residue, which corresponds to R223 in GcvT, is not conserved in SesA, LigM, and DesA. We predict that the amino acid residues act as proton donors that provide the methylenedioxy bridges of sesamin with a proton. Considering the crystal structures of GcvT, DmdA, and DMGO in the GcvT family proteins, candidates of proton donor residues in the predicted active site of SesA are as follows: R81, H82, R100, R179, and H225 (SI Appendix, Fig. S38). BH⁺ and B′H⁺ provide protons and become :B and :B′, respectively, in sesamin. In the proposed reaction (Fig. 4C), the ring closure to yield 5,10-CH₂-THF is predicted to be initiated by a base (:B′). Then, the :B′ should accept the proton on the N10 atom of an iminium ion, including the N5 atom of 5-CH₂-THF in the last step (step iii). The SesA reaction is different from that of other GcvT family enzymes in that SesA requires proton donors for the reaction. To identify the amino acid residues B and B′, studies on the crystal structure and site-directed mutagenesis studies of SesA are required.

At the beginning of this study, we isolated strain no. 22 by enrichment culture using sesamin as a sole-carbon source. In the growth experiment, the enzymatic activity of SesA was found to increase just before the onset of cell growth (SI Appendix, Fig. S7). These experiments and Western-blot analysis revealed that SesA formation was induced by sesamin in both media. Moreover, 5,10-CH₂-THF produced by the SesA reaction would be metabolized as follows. Analysis of the gene annotation of strain no. 22 indicated that strain no. 22 has a putative formyltetrahydrofolate deformylase gene (thf1), and a putative bifunctional 5,10-CH₂-THF dehydrogenase/cyclohydrolase gene (thf2) upstream and downstream of the sesA gene, respectively (Fig. 4B). Thf2 could convert 5,10-CH₂-THF into 10-formyltetrahydrofolate (10-CHO-THF), which is a substrate for the biosynthesis of purine. Also, Thf1 could convert 10-CHO-THF into THF. These findings suggest that strain in sesamin no. 22 is important physiologically.

Some SesA homologs, which we found by Blast searches, form a gene cluster with folate-metabolizing enzyme genes (SI Appendix, Fig. S39). Most were derived from Actinobacteria, but this gene set was also observed in the Gram-negative bacterium, Bradyrhizobium japonicum WSM2793. These findings suggest that THF-dependent C1 transferases are distributed in various microorganisms.

Materials and Methods

Bacterial Strains, Plasmids, Primers, and Additional Methods.

For bacterial strains, plasmids, and primers, see SI Appendix, Tables S1 and S2.

For chemicals, HPLC and LC/MS/MS analyses, structure determination, purification of the sesamin-metabolizing enzyme from Sinomonas sp. no. 22, the draft genome sequence of Sinomonas sp. no. 22, cloning and heterologous expression of sesA, determination of the molecular mass of SesA, time courses of cell growth and enzymatic activity, Western blot analysis, measurement of folate, temperature dependency and stability, pH dependency and stability, substrate specificity, circular dichroism analysis, and site-directed mutagenesis, see SI Appendix, Supplementary Methods.

Isolation of Sesamin-Metabolizing Microorganisms.

Sesamin-metabolizing microorganisms were isolated from soil in the University of Tsukuba and sesame gardens by the following enrichment method. Step 1 was as follows: 1 g of collected soil was added to 10 mL of sesamin medium, which consisted of 0.1% (wt/vol) sesamin, 1% (wt/vol) (NH₄)₂SO₄, 0.05% (wt/vol) KH₂PO₄, 0.05% (wt/vol) K₂HPO₄, 0.05% (wt/vol) MgSO₄·7H₂O, 0.0005% (wt/vol) FeSO₄·7H₂O, and 10% (vol/vol) tap water, adjusted to pH 7.0 with NaOH, followed by incubation at 28 °C or 37 °C for 3 d. Step 2 was as follows: 2% (vol/vol) of the cultivated medium was added to the same fresh medium, followed by incubation at 28 °C or 37 °C for 3 d. Step 2 was repeated three times.

After enrichment, the culture broth was spread on sesamin sole-carbon agar plates, which contained 1.5% (wt/vol) agar in addition to the above sesamin sole-carbon medium, and colonies that grew on these plates on 1 wk incubation at 28 °C were isolated.

Each of the isolated strains was inoculated into a test tube containing 10 mL of sesamin sole-carbon medium, followed by incubation at 28 °C for 2 d. Cells were harvested by centrifugation (4,000 × g, 10 min, 4 °C) and, after washing twice with 10 mM potassium phosphate buffer (KPB) (pH 7.0), were resuspended in 200 μL of the same buffer. Then, the cells were disrupted by sonication, and the cell debris was removed by centrifugation (27,000 × g, 10 min, 4 °C) to prepare a cell-free extract. Two hundred microliters of the reaction mixture comprised 10 μL of 100 mM KPB (pH 7.0), 10 μL of 10 mM sesamin (in DMSO), 100 μL of the cell-free extract, and milliQ water. After incubation at 28 °C for 16 h, the reaction was stopped by adding 100 μL of acetonitrile. The reaction samples were analyzed by HPLC and LC/MS.

Enzyme Assay.

Measurement of enzyme activity was performed as follows. One hundred microliters of the reaction mixture [1 μL of 0.73 mg/mL SesA, 5 μL of 1 M Tris⋅HCl (pH 8.0), 3 μL of 10 mM substrate (in DMSO), 10 μL of 10 mM THF, and 2 μL of Tween 80 were used]. THF was dissolved in 50 mM Tris⋅HCl (pH 9.0), 1% 2-mercaptoethanol, and 2% (wt/vol) ascorbate. One unit of sesamin-converting activity was defined as the amount of enzyme required to catalyze the formation of 1 μmol of sesamin monocatechol per minute. Specific activity is expressed as units per milligram of protein.

The reaction was initiated by adding the enzyme, followed by incubation at 28 °C for an appropriate time. After incubation, the reaction was stopped by adding 100 μL of acetonitrile.

For determination of the kinetic parameters for the demethylenation of sesamin, 100 μL of the reaction mixture consists of 1 μL of 0.0731 mg/mL SesA, 5 μL of 1 M Tris⋅HCl (pH 8.0), 10 μL of 10 mM THF, 2 μL of Tween 80, and from 0.05 mM to 0.3 mM sesamin. The reactions were initiated by the addition of SesA, followed by incubation at 28 °C, and then termination at 1, 3, 5, 7, and 10 min by the addition of 50 μL of acetonitrile. The experiments were carried out in duplicate independently. The k_cat values were calculated using a M_r of 50,385 for SesA.

Nucleotide Sequence Accession Numbers.

The nucleotide sequence data reported in this paper appear in the DDBJ/GenBank database under accession numbers LC101493 for sesA and thf2, LC101494 for thf1, and LC101495 for the 16S rRNA gene.

Acknowledgments

We thank Dr. Kentaro Shiraki (University of Tsukuba) for help with circular dichroism spectra analysis.

Footnotes

↵¹T.K. and E.F. contributed equally to this work.
↵²To whom correspondence should be addressed. Email: kobay{at}agbi.tsukuba.ac.jp.

Author contributions: T.K., E.F., and M.K. designed research; T.K. and E.F. performed research; T.K., E.F., Y.H., and M.K. analyzed data; and T.K. and M.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The nucleotide sequence data reported in this paper have been deposited in the DNA Data Bank of Japan (DDBJ) database [accession nos. LC101493 (sesA and thf2), LC101494 (thf1), and LC101495 (16S rRNA gene)].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1605050113/-/DCSupplemental.

References

↵
1. Kobayashi M,
2. Shimizu S
(1998) Metalloenzyme nitrile hydratase: Structure, regulation, and application to biotechnology. Nat Biotechnol 16(8):733–736.
.
OpenUrl CrossRef PubMed
↵
1. Komeda H,
2. Hori Y,
3. Kobayashi M,
4. Shimizu S
(1996) Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase. Proc Natl Acad Sci USA 93(20):10572–10577.
.
OpenUrl Abstract/FREE Full Text
↵
1. Nomura J, et al.
(2013) Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis. Proc Natl Acad Sci USA 110(8):2810–2815.
.
OpenUrl Abstract/FREE Full Text
↵
1. Hassaninasab A,
2. Hashimoto Y,
3. Tomita-Yokotani K,
4. Kobayashi M
(2011) Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc Natl Acad Sci USA 108(16):6615–6620.
.
OpenUrl Abstract/FREE Full Text
↵
1. Hearon W-M,
2. MacGregor W-S
(1955) The naturally occurring lignans. Chem Rev 55(5):957–1067.
.
OpenUrl CrossRef
↵
1. Umezawa T
(2003) Diversity in lignan biosynthesis. Phytochem Rev 2(3):371–390.
.
OpenUrl CrossRef
↵
1. Fukuda Y,
2. Nagata M,
3. Osawa T,
4. Namiki M
(1986) Chemical aspects of the antioxidative activity of roasted sesame seed oil, and the effect of using the oil for frying. Agric Biol Chem 50(4):857–862.
.
OpenUrl
↵
1. Hirose N, et al.
(1991) Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 32(4):629–638.
.
OpenUrl Abstract
↵
1. Tsuruoka N,
2. Kidokoro A,
3. Matsumoto I,
4. Abe K,
5. Kiso Y
(2005) Modulating effect of sesamin, a functional lignan in sesame seeds, on the transcription levels of lipid- and alcohol-metabolizing enzymes in rat liver: A DNA microarray study. Biosci Biotechnol Biochem 69(1):179–188.
.
OpenUrl CrossRef PubMed
↵
1. Miyawaki T, et al.
(2009) Antihypertensive effects of sesamin in humans. J Nutr Sci Vitaminol (Tokyo) 55(1):87–91.
.
OpenUrl CrossRef PubMed
↵
1. Utsunomiya T,
2. Chavali S-R,
3. Zhong W-W,
4. Forse R-A
(2000) Effects of sesamin-supplemented dietary fat emulsions on the ex vivo production of lipopolysaccharide-induced prostanoids and tumor necrosis factor alpha in rats. Am J Clin Nutr 72(3):804–808.
.
OpenUrl Abstract/FREE Full Text
↵
1. Yasuda K,
2. Ikushiro S,
3. Kamakura M,
4. Ohta M,
5. Sakaki T
(2010) Metabolism of sesamin by cytochrome P450 in human liver microsomes. Drug Metab Dispos 38(12):2117–2123.
.
OpenUrl Abstract/FREE Full Text
↵
1. Sakaki T, et al.
(2012) Metabolism of sesamin by CYPs and UGTs in human liver. J Food Drug Anal 20(1):376–379.
.
OpenUrl
↵
1. Moazzami A-A,
2. Andersson R-E,
3. Kamal-Eldin A
(2007) Quantitative NMR analysis of a sesamin catechol metabolite in human urine. J Nutr 137(4):940–944.
.
OpenUrl Abstract/FREE Full Text
↵
1. Miyake Y, et al.
(2005) Antioxidative catechol lignans converted from sesamin and sesaminol triglucoside by culturing with Aspergillus. J Agric Food Chem 53(1):22–27.
.
OpenUrl CrossRef PubMed
↵
1. Liu Z,
2. Saarinen N-M,
3. Thompson L-U
(2006) Sesamin is one of the major precursors of mammalian lignans in sesame seed (Sesamum indicum) as observed in vitro and in rats. J Nutr 136(4):906–912.
.
OpenUrl Abstract/FREE Full Text
↵
1. Kiso Y
(2004) Antioxidative roles of sesamin, a functional lignan in sesame seed, and it’s effect on lipid- and alcohol-metabolism in the liver: A DNA microarray study. Biofactors 21(1-4):191–196.
.
OpenUrl CrossRef PubMed
↵
1. Prasad K
(2000) Antioxidant activity of secoisolariciresinol diglucoside-derived metabolites, secoisolariciresinol, enterodiol, and enterolactone. Int J Angiol 9(4):220–225.
.
OpenUrl CrossRef PubMed
↵
1. Peterson J, et al.
(2010) Dietary lignans: Physiology and potential for cardiovascular disease risk reduction. Nutr Rev 68(10):571–603.
.
OpenUrl Abstract/FREE Full Text
↵
1. Cunnane S-C, et al.
(1995) Nutritional attributes of traditional flaxseed in healthy young adults. Am J Clin Nutr 61(1):62–68.
.
OpenUrl Abstract/FREE Full Text
↵
1. Hallund J,
2. Tetens I,
3. Bügel S,
4. Tholstrup T,
5. Bruun J-M
(2008) The effect of a lignan complex isolated from flaxseed on inflammation markers in healthy postmenopausal women. Nutr Metab Cardiovasc Dis 18(7):497–502.
.
OpenUrl CrossRef PubMed
↵
1. Nakai M, et al.
(2003) Novel antioxidative metabolites in rat liver with ingested sesamin. J Agric Food Chem 51(6):1666–1670.
.
OpenUrl CrossRef PubMed
↵
1. Leys D,
2. Basran J,
3. Scrutton N-S
(2003) Channelling and formation of ‘active’ formaldehyde in dimethylglycine oxidase. EMBO J 22(16):4038–4048.
.
OpenUrl Abstract/FREE Full Text
↵
1. Ida K,
2. Moriguchi T,
3. Suzuki H
(2005) Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96. Biochem Biophys Res Commun 333(2):359–366.
.
OpenUrl CrossRef PubMed
↵
1. Schuller D-J,
2. Reisch C-R,
3. Moran M-A,
4. Whitman W-B,
5. Lanzilotta W-N
(2012) Structures of dimethylsulfoniopropionate-dependent demethylase from the marine organism Pelagabacter ubique. Protein Sci 21(2):289–298.
.
OpenUrl CrossRef PubMed
↵
1. Nishikawa S, et al.
(1998) Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate. Appl Environ Microbiol 64(3):836–842.
.
OpenUrl Abstract/FREE Full Text
↵
1. Masai E, et al.
(2004) A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate. J Bacteriol 186(9):2757–2765.
.
OpenUrl Abstract/FREE Full Text
↵
1. Abe T,
2. Masai E,
3. Miyauchi K,
4. Katayama Y,
5. Fukuda M
(2005) A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6. J Bacteriol 187(6):2030–2037.
.
OpenUrl Abstract/FREE Full Text
↵
1. Lee H-H,
2. Kim D-J,
3. Ahn H-J,
4. Ha J-Y,
5. Suh S-W
(2004) Crystal structure of T-protein of the glycine cleavage system: Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia. J Biol Chem 279(48):50514–50523.
.
OpenUrl Abstract/FREE Full Text
↵
1. Hiratsuka T, et al.
(2008) An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321(5896):1670–1673.
.
OpenUrl Abstract/FREE Full Text
↵
1. Ahmad M, et al.
(2011) Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 50(23):5096–5107.
.
OpenUrl CrossRef PubMed
↵
1. Van der Geize R, et al.
(2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci USA 104(6):1947–1952.
.
OpenUrl Abstract/FREE Full Text
↵
1. Marín L,
2. Miguélez E-M,
3. Villar C-J,
4. Lombó F
(2015) Bioavailability of dietary polyphenols and gut microbiota metabolism: antimicrobial properties. BioMed Res Int 2015:905215.
.
OpenUrl PubMed
↵
1. Nishiyama T,
2. Hashimoto Y,
3. Kusakabe H,
4. Kumano T,
5. Kobayashi M
(2014) Natural low-molecular mass organic compounds with oxidase activity as organocatalysts. Proc Natl Acad Sci USA 111(48):17152–17157.
.
OpenUrl Abstract/FREE Full Text
↵
1. Tucker G-T, et al.
(1994) The demethylenation of methylenedioxymethamphetamine (“ecstasy”) by debrisoquine hydroxylase (CYP2D6). Biochem Pharmacol 47(7):1151–1156.
.
OpenUrl CrossRef PubMed
↵
1. Buswell J-A,
2. Cain R-B
(1973) Microbial degradation of piperonylic acid by Pseudomonas fluorescens. FEBS Lett 29(3):297–300.
.
OpenUrl CrossRef PubMed
↵
1. Hasse D, et al.
(2013) Structure of the homodimeric glycine decarboxylase P-protein from Synechocystis sp. PCC 6803 suggests a mechanism for redox regulation. J Biol Chem 288(49):35333–35345.
.
OpenUrl Abstract/FREE Full Text
↵
1. Okamura-Ikeda K, et al.
(2010) Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: Implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism. J Biol Chem 285(24):18684–18692.
.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 113 (32)

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Article Classifications

Biological Sciences
Microbiology

[1] ↵
Kobayashi M,
Shimizu S
(1998) Metalloenzyme nitrile hydratase: Structure, regulation, and application to biotechnology. Nat Biotechnol 16(8):733–736.
.
OpenUrl CrossRef PubMed

[2] Kobayashi M,

[3] Shimizu S

[4] ↵
Komeda H,
Hori Y,
Kobayashi M,
Shimizu S
(1996) Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase. Proc Natl Acad Sci USA 93(20):10572–10577.
.
OpenUrl Abstract/FREE Full Text

[5] Komeda H,

[6] Hori Y,

[7] Kobayashi M,

[8] Shimizu S

[9] ↵
Nomura J, et al.
(2013) Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis. Proc Natl Acad Sci USA 110(8):2810–2815.
.
OpenUrl Abstract/FREE Full Text

[10] Nomura J, et al.

[11] ↵
Hassaninasab A,
Hashimoto Y,
Tomita-Yokotani K,
Kobayashi M
(2011) Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc Natl Acad Sci USA 108(16):6615–6620.
.
OpenUrl Abstract/FREE Full Text

[12] Hassaninasab A,

[13] Hashimoto Y,

[14] Tomita-Yokotani K,

[15] Kobayashi M

[16] ↵
Hearon W-M,
MacGregor W-S
(1955) The naturally occurring lignans. Chem Rev 55(5):957–1067.
.
OpenUrl CrossRef

[17] Hearon W-M,

[18] MacGregor W-S

[19] ↵
Umezawa T
(2003) Diversity in lignan biosynthesis. Phytochem Rev 2(3):371–390.
.
OpenUrl CrossRef

[20] Umezawa T

[21] ↵
Fukuda Y,
Nagata M,
Osawa T,
Namiki M
(1986) Chemical aspects of the antioxidative activity of roasted sesame seed oil, and the effect of using the oil for frying. Agric Biol Chem 50(4):857–862.
.
OpenUrl

[22] Fukuda Y,

[23] Nagata M,

[24] Osawa T,

[25] Namiki M

[26] ↵
Hirose N, et al.
(1991) Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 32(4):629–638.
.
OpenUrl Abstract

[27] Hirose N, et al.

[28] ↵
Tsuruoka N,
Kidokoro A,
Matsumoto I,
Abe K,
Kiso Y
(2005) Modulating effect of sesamin, a functional lignan in sesame seeds, on the transcription levels of lipid- and alcohol-metabolizing enzymes in rat liver: A DNA microarray study. Biosci Biotechnol Biochem 69(1):179–188.
.
OpenUrl CrossRef PubMed

[29] Tsuruoka N,

[30] Kidokoro A,

[31] Matsumoto I,

[32] Abe K,

[33] Kiso Y

[34] ↵
Miyawaki T, et al.
(2009) Antihypertensive effects of sesamin in humans. J Nutr Sci Vitaminol (Tokyo) 55(1):87–91.
.
OpenUrl CrossRef PubMed

[35] Miyawaki T, et al.

[36] ↵
Utsunomiya T,
Chavali S-R,
Zhong W-W,
Forse R-A
(2000) Effects of sesamin-supplemented dietary fat emulsions on the ex vivo production of lipopolysaccharide-induced prostanoids and tumor necrosis factor alpha in rats. Am J Clin Nutr 72(3):804–808.
.
OpenUrl Abstract/FREE Full Text

[37] Utsunomiya T,

[38] Chavali S-R,

[39] Zhong W-W,

[40] Forse R-A

[41] ↵
Yasuda K,
Ikushiro S,
Kamakura M,
Ohta M,
Sakaki T
(2010) Metabolism of sesamin by cytochrome P450 in human liver microsomes. Drug Metab Dispos 38(12):2117–2123.
.
OpenUrl Abstract/FREE Full Text

[42] Yasuda K,

[43] Ikushiro S,

[44] Kamakura M,

[45] Ohta M,

[46] Sakaki T

[47] ↵
Sakaki T, et al.
(2012) Metabolism of sesamin by CYPs and UGTs in human liver. J Food Drug Anal 20(1):376–379.
.
OpenUrl

[48] Sakaki T, et al.

[49] ↵
Moazzami A-A,
Andersson R-E,
Kamal-Eldin A
(2007) Quantitative NMR analysis of a sesamin catechol metabolite in human urine. J Nutr 137(4):940–944.
.
OpenUrl Abstract/FREE Full Text

[50] Moazzami A-A,

[51] Andersson R-E,

[52] Kamal-Eldin A

[53] ↵
Miyake Y, et al.
(2005) Antioxidative catechol lignans converted from sesamin and sesaminol triglucoside by culturing with Aspergillus. J Agric Food Chem 53(1):22–27.
.
OpenUrl CrossRef PubMed

[54] Miyake Y, et al.

[55] ↵
Liu Z,
Saarinen N-M,
Thompson L-U
(2006) Sesamin is one of the major precursors of mammalian lignans in sesame seed (Sesamum indicum) as observed in vitro and in rats. J Nutr 136(4):906–912.
.
OpenUrl Abstract/FREE Full Text

[56] Liu Z,

[57] Saarinen N-M,

[58] Thompson L-U

[59] ↵
Kiso Y
(2004) Antioxidative roles of sesamin, a functional lignan in sesame seed, and it’s effect on lipid- and alcohol-metabolism in the liver: A DNA microarray study. Biofactors 21(1-4):191–196.
.
OpenUrl CrossRef PubMed

[60] Kiso Y

[61] ↵
Prasad K
(2000) Antioxidant activity of secoisolariciresinol diglucoside-derived metabolites, secoisolariciresinol, enterodiol, and enterolactone. Int J Angiol 9(4):220–225.
.
OpenUrl CrossRef PubMed

[62] Prasad K

[63] ↵
Peterson J, et al.
(2010) Dietary lignans: Physiology and potential for cardiovascular disease risk reduction. Nutr Rev 68(10):571–603.
.
OpenUrl Abstract/FREE Full Text

[64] Peterson J, et al.

[65] ↵
Cunnane S-C, et al.
(1995) Nutritional attributes of traditional flaxseed in healthy young adults. Am J Clin Nutr 61(1):62–68.
.
OpenUrl Abstract/FREE Full Text

[66] Cunnane S-C, et al.

[67] ↵
Hallund J,
Tetens I,
Bügel S,
Tholstrup T,
Bruun J-M
(2008) The effect of a lignan complex isolated from flaxseed on inflammation markers in healthy postmenopausal women. Nutr Metab Cardiovasc Dis 18(7):497–502.
.
OpenUrl CrossRef PubMed

[68] Hallund J,

[69] Tetens I,

[70] Bügel S,

[71] Tholstrup T,

[72] Bruun J-M

[73] ↵
Nakai M, et al.
(2003) Novel antioxidative metabolites in rat liver with ingested sesamin. J Agric Food Chem 51(6):1666–1670.
.
OpenUrl CrossRef PubMed

[74] Nakai M, et al.

[75] ↵
Leys D,
Basran J,
Scrutton N-S
(2003) Channelling and formation of ‘active’ formaldehyde in dimethylglycine oxidase. EMBO J 22(16):4038–4048.
.
OpenUrl Abstract/FREE Full Text

[76] Leys D,

[77] Basran J,

[78] Scrutton N-S

[79] ↵
Ida K,
Moriguchi T,
Suzuki H
(2005) Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96. Biochem Biophys Res Commun 333(2):359–366.
.
OpenUrl CrossRef PubMed

[80] Ida K,

[81] Moriguchi T,

[82] Suzuki H

[83] ↵
Schuller D-J,
Reisch C-R,
Moran M-A,
Whitman W-B,
Lanzilotta W-N
(2012) Structures of dimethylsulfoniopropionate-dependent demethylase from the marine organism Pelagabacter ubique. Protein Sci 21(2):289–298.
.
OpenUrl CrossRef PubMed

[84] Schuller D-J,

[85] Reisch C-R,

[86] Moran M-A,

[87] Whitman W-B,

[88] Lanzilotta W-N

[89] ↵
Nishikawa S, et al.
(1998) Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate. Appl Environ Microbiol 64(3):836–842.
.
OpenUrl Abstract/FREE Full Text

[90] Nishikawa S, et al.

[91] ↵
Masai E, et al.
(2004) A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate. J Bacteriol 186(9):2757–2765.
.
OpenUrl Abstract/FREE Full Text

[92] Masai E, et al.

[93] ↵
Abe T,
Masai E,
Miyauchi K,
Katayama Y,
Fukuda M
(2005) A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6. J Bacteriol 187(6):2030–2037.
.
OpenUrl Abstract/FREE Full Text

[94] Abe T,

[95] Masai E,

[96] Miyauchi K,

[97] Katayama Y,

[98] Fukuda M

[99] ↵
Lee H-H,
Kim D-J,
Ahn H-J,
Ha J-Y,
Suh S-W
(2004) Crystal structure of T-protein of the glycine cleavage system: Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia. J Biol Chem 279(48):50514–50523.
.
OpenUrl Abstract/FREE Full Text

[100] Lee H-H,

[101] Kim D-J,

[102] Ahn H-J,

[103] Ha J-Y,

[104] Suh S-W

[105] ↵
Hiratsuka T, et al.
(2008) An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321(5896):1670–1673.
.
OpenUrl Abstract/FREE Full Text

[106] Hiratsuka T, et al.

[107] ↵
Ahmad M, et al.
(2011) Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 50(23):5096–5107.
.
OpenUrl CrossRef PubMed

[108] Ahmad M, et al.

[109] ↵
Van der Geize R, et al.
(2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci USA 104(6):1947–1952.
.
OpenUrl Abstract/FREE Full Text

[110] Van der Geize R, et al.

[111] ↵
Marín L,
Miguélez E-M,
Villar C-J,
Lombó F
(2015) Bioavailability of dietary polyphenols and gut microbiota metabolism: antimicrobial properties. BioMed Res Int 2015:905215.
.
OpenUrl PubMed

[112] Marín L,

[113] Miguélez E-M,

[114] Villar C-J,

[115] Lombó F

[116] ↵
Nishiyama T,
Hashimoto Y,
Kusakabe H,
Kumano T,
Kobayashi M
(2014) Natural low-molecular mass organic compounds with oxidase activity as organocatalysts. Proc Natl Acad Sci USA 111(48):17152–17157.
.
OpenUrl Abstract/FREE Full Text

[117] Nishiyama T,

[118] Hashimoto Y,

[119] Kusakabe H,

[120] Kumano T,

[121] Kobayashi M

[122] ↵
Tucker G-T, et al.
(1994) The demethylenation of methylenedioxymethamphetamine (“ecstasy”) by debrisoquine hydroxylase (CYP2D6). Biochem Pharmacol 47(7):1151–1156.
.
OpenUrl CrossRef PubMed

[123] Tucker G-T, et al.

[124] ↵
Buswell J-A,
Cain R-B
(1973) Microbial degradation of piperonylic acid by Pseudomonas fluorescens. FEBS Lett 29(3):297–300.
.
OpenUrl CrossRef PubMed

[125] Buswell J-A,

[126] Cain R-B

[127] ↵
Hasse D, et al.
(2013) Structure of the homodimeric glycine decarboxylase P-protein from Synechocystis sp. PCC 6803 suggests a mechanism for redox regulation. J Biol Chem 288(49):35333–35345.
.
OpenUrl Abstract/FREE Full Text

[128] Hasse D, et al.

[129] ↵
Okamura-Ikeda K, et al.
(2010) Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: Implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism. J Biol Chem 285(24):18684–18692.
.
OpenUrl Abstract/FREE Full Text

[130] Okamura-Ikeda K, et al.

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New Research In

Discovery of a sesamin-metabolizing microorganism and a new enzyme

Significance

Abstract

Results

Isolation and Identification of Sesamin-Metabolizing Bacteria.

Structure Determination of the Sesamin Metabolites.

Purification of the Sesamin-Metabolizing Enzyme.

Identification of the Sesamin-Metabolizing Enzyme.

Time-Dependent Sesamin Metabolism During Cultivation.

Western Blot Analysis.

Cloning and Heterologous Expression of sesA and Biochemical Properties of SesA.

Stoichiometry.

Kinetic Analysis.

Substrate Specificity.

Mutational Analysis of SesA.

Discussion

Materials and Methods

Bacterial Strains, Plasmids, Primers, and Additional Methods.

Isolation of Sesamin-Metabolizing Microorganisms.

Enzyme Assay.

Nucleotide Sequence Accession Numbers.

Acknowledgments

Footnotes

References

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Discovery of a sesamin-metabolizing microorganism and a new enzyme

Significance

Abstract

Results

Isolation and Identification of Sesamin-Metabolizing Bacteria.

Structure Determination of the Sesamin Metabolites.

Purification of the Sesamin-Metabolizing Enzyme.

Identification of the Sesamin-Metabolizing Enzyme.

Time-Dependent Sesamin Metabolism During Cultivation.

Western Blot Analysis.

Cloning and Heterologous Expression of sesA and Biochemical Properties of SesA.

Stoichiometry.

Kinetic Analysis.

Substrate Specificity.

Mutational Analysis of SesA.

Discussion

Materials and Methods

Bacterial Strains, Plasmids, Primers, and Additional Methods.

Isolation of Sesamin-Metabolizing Microorganisms.

Enzyme Assay.

Nucleotide Sequence Accession Numbers.

Acknowledgments

Footnotes

References

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