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Home , Aminomethyltransferase, Bifunctional, Dimethylglycine oxidase, Methylenedioxy, Sarcosine oxidase

Discovery of a sesamin-metabolizing microorganism and a new

Takuto Kumanoa,1, Etsuko Fujikia,1, Yoshiteru Hashimotoa, and Michihiko Kobayashia,2

aGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Edited by Julian Davies, University of British Columbia, Vancouver, BC, Canada, and approved June 14, 2016 (received for review March 28, 2016) Sesamin is one of the major lignans found in sesame oil. Although higher antioxidant activity than vitamin E (18); reduction of some microbial metabolites of sesamin have been identified, cardiovascular disease risk (19); reduction of the risk of arte- sesamin-metabolic pathways remain uncharacterized at both the riosclerosis by decreasing the total cholesterol level (20); and enzyme and gene levels. Here, we isolated microorganisms growing reduction of inflammation markers (21). However, neither ses- on sesamin as a sole- source. One microorganism showing amin-metabolizing nor their genes have been identified Sinomonas significant sesamin-degrading activity was identified as in microorganisms, including intestinal bacteria and A. oryzae. sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified Here, we describe the isolation and identification of a sesamin- from this strain and characterized. SesA catalyzed catabolizing soil microorganism, Sinomonas sp. no. 22, together transfer from sesamin or sesamin monocatechol to tetrahydrofolate with purification and characterization of a previously un- (THF) with ring cleavage, yielding sesamin mono- or di-catechol and known sesamin-modifying enzyme. This enzyme has the unique 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were catalytic ability to transfer the methylene group from sesamin to determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, −1 −1 −1 tetrahydrofolate (THF) through ring cleavage (Fig. 1). In addition, Vmax = 9.3 ± 0.4 (μmol·min ·mg ), and kcat = 7.9 ± 0.3 s . Next, we investigated the specificity. SesA also showed enzymatic we clarify the biochemical properties of this enzyme and propose a activity toward (+)-episesamin, (−)-asarinin, sesaminol, (+)-sesamolin, possible reaction mechanism. Our findings provide novel insights and piperine. Growth studies with strain no. 22, and Western blot into the catabolism of natural compounds and THF-dependent analysis revealed that SesA formation is inducible by sesamin. The metabolic pathways. deduced amino acid sequence of sesA exhibited weak overall se- MICROBIOLOGY quence similarity to that of the family of cleavage Results T- (GcvTs), which catalyze glycine degradation in most Isolation and Identification of Sesamin-Metabolizing Bacteria. The bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 trans- soil samples used for microbial screening were collected from fer to THF with ring cleavage reaction among GcvT family proteins. sesame gardens at the University of Tsukuba. At ∼1 mo from the Moreover, SesA homolog genes are found in both Gram-positive start of this study, by using the enrichment culture method de- and Gram-negative bacteria. Our findings provide new insights scribed in Materials and Methods, we isolated 40 microorganisms into microbial sesamin metabolism and the function of GcvT that were able to grow on culture medium containing sesamin as family proteins. the sole carbon source. We selected one isolate, strain no. 22, for further study. This strain showed 99% 16S rRNA gene sequence sesamin | metabolism | lignan | tetrahydrofolate T similarity to Sinomonas atrocyanea DSM20127 . Morphological and biochemical properties of strain no. 22 are shown in SI Ap- e have been involved in studies of not only microbial pendix, Supplementary Data. Wmetabolism of man-made compounds (1–3) but also bio- logically active natural compounds, such as curcumin (4). In this Significance study, we characterize the microbial metabolism of the lignan sesamin. Lignans (5) are plant-derived compounds consisting of dimers Lignans, including sesamin, are produced by a wide variety of plants, but the microbial degradation of lignan has not been of phenylpropane units (6). They are found in a wide variety of Sinomonas plant-based foods. Whole-grain products, vegetables, fruits, nuts, identified biochemically. Here, we show that sp. no. seeds, and beverages such as tea, coffee, and wine are dietary 22 can catabolize sesamin as a sole-carbon source. We identi- fied the sesamin-converting enzyme, SesA, from strain Sino- sources of lignans. In Asian countries, sesame, which contains monas sp. no. 22. SesA catalyzed methylene group transfer lignans, is used traditionally as a food. A major lignan is sesamin, from sesamin to tetrahydrofolate (THF). The resulting 5,10-CH - which is a biologically active compound with antioxidative (7), 2 THF might find use as a C1-donor for bioprocesses. SesA gene cholesterol-lowering (8), lipid-lowering (9), antihypertensive (10), homologs were found in the genomes of both Gram-positive and antiinflammatory (11) properties. and Gram-negative bacteria, suggesting that sesamin (lignan) In humans, sesamin is metabolized by CYP450 enzymes into utilization is a widespread, but still unrecognized, function in sesamin mono- and di-catechol in liver microsomes (12). Sesamin environments where lignans are produced and degraded. monocatechol is metabolized further by UDP-glucuronosyltrans-

ferase (UGT) and O-methyl (COMT) (13), and the Author contributions: T.K., E.F., and M.K. designed research; T.K. and E.F. performed resulting glucuronides of sesamin metabolites are excreted in the research; T.K., E.F., Y.H., and M.K. analyzed data; and T.K. and M.K. wrote the paper. bile and urine (14). The authors declare no conflict of interest. In microorganisms, on the other hand, metabolism of sesamin This article is a PNAS Direct Submission. has been reported in few species. Aspergillus oryzae converts Data deposition: The nucleotide sequence data reported in this paper have been depos- sesamin to sesamin mono- and di-catechol (15) whereas in- ited in the DNA Data Bank of Japan (DDBJ) database [accession nos. LC101493 (sesA and testinal bacteria convert sesamin to the so-called “mammalian thf2), LC101494 (thf1), and LC101495 (16S rRNA gene)]. lignans” enterodiol and enterolactone (16). The activities of 1T.K. and E.F. contributed equally to this work. these metabolites make them useful dietary substances. Sesamin 2To whom correspondence should be addressed. Email: [email protected]. mono- and di-catechol show stronger antioxidant activity than This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sesamin (17). Also, mammalian lignans show other properties: 1073/pnas.1605050113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1605050113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 ABTime-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 Fig. 1. Schematic representation of the bond cleavage location in the × structure of substrates for SesA (A) and previously reported THF-dependent activity was not observed during cultivation. Furthermore, in 2 methyl (B). The cleavage site of the bond between within YT liquid medium, sesamin-converting activity was detected only a substrate is indicated by a dashed line. in the presence of added sesamin (SI Appendix,Fig.S7B).

Western Blot Analysis. To confirm correlation of SesA expression Structure Determination of the Sesamin Metabolites. We incubated with sesamin metabolism, the induction of SesA in the presence a cell-free extract of strain no. 22 with sesamin as a substrate. of sesamin was followed by Western blot analysis on cell-free Two products (compounds A and B) were isolated by ethyl ac- extracts in the following culture conditions: One was in 0.1% (wt/vol) etate extraction and preparative HPLC (Fig. 2). sesamin-supplemented 2× YT liquid medium, and the other was in High-resolution mass spectrometry (HRMS) analysis in the in 2× YT liquid medium. A cross-reacting band was observed negative mode revealed the molecular ion of compound A at m/z − only in the cell-free extract of cells cultured with sesamin (SI Ap- 341.1029 [M-H] , which was in agreement with the calculated pendix,Fig.S7D). In addition, SesA enzyme activity (0.053 units/mg) mass of C19H17O6, and that of compound B at m/z 329.1022 − wasobservedonlyincellsgrowninthepresenceofsesamin. [M-H] , in agreement with the calculated mass of C18H17O6 (SI Appendix, Fig. S1). The loss of 12 atomic mass units suggested Cloning and Heterologous Expression of sesA and Biochemical modification of the methylenedioxyphenyl group. Properties of SesA. The 1.4-kb region of the SesA-coding gene 1H NMR spectra of compounds A and B showed proton signals was inserted into an expression vector, and recombinant SesA corresponding to the tetrahydrofuran ring and 1,3,4-substituted was produced in Escherichia coli and purified. The specific ac- moiety expressed (SI Appendix, Fig. S1). On the other tivity of the recombinant SesA was approximately the same hand, the proton signal corresponding to H-10′ of sesamin was (8.8 units/mg) as that of SesA purified from strain no. 22. absent in compound A (SI Appendix, Figs. S1 and S2). The proton We examined the effects of temperature and pH on SesA signals corresponding to H-10 and H-10′ of sesamin were not activity. The optimal reaction temperature and pH were below observed for compound B (SI Appendix, Figs. S1 and S3). 40 °C and pH 7.5–8.5, respectively (SI Appendix, Fig. S8 A and B). Based on these observations, compounds A and B were iden- SesA was most stable under 30 °C and in the pH range of 5.5–10.0 tified as sesamin monocatechol (1 in Fig. 3) and sesamin di- (SI Appendix,Fig.S8C and D). catechol (2 in Fig. 3), respectively (22). The absorption spectrum of the purified SesA showed an ab- sorbance maximum near 280 nm. No other absorption peak or Purification of the Sesamin-Metabolizing Enzyme. The sesamin- shoulder was observed at higher wavelengths (SI Appendix, Fig. metabolizing enzyme was purified 1.2-fold with a yield of 3.4% S9). These results suggest that no is bound to the pu- (SI Appendix, Table S3). Considering the relationship between rified enzyme. The CD spectrum is shown in SI Appendix, Fig. the protein amount and the activity of each fraction obtained S10. Qualitative analysis of metal content was performed by in- on gel-filtration chromatography, the target enzyme was as the ductively coupled plasma atomic emission analysis. ∼50-kDa protein band observed on SDS/PAGE (SI Appendix, The enzyme contained 1.70 mol of phosphorus and 0.70 mol of Fig. S4 and Table S4). An N-terminal amino acid sequence of sulfur per mole of subunit. No other metal was detected within this protein was determined to be TAEQAIN. the assay limits (SI Appendix, Supplementary Data 3). The gene for the sesamin-metabolizing enzyme, named SesA, was identified from the draft genome sequence data for strain Stoichiometry. The stoichiometry of the SesA reaction was exam- no. 22 (see Identification of the Sesamin-Metabolizing Enzyme). ined. The amounts of sesamin, sesamin monocatechol, sesamin The primary structure of SesA suggested that it might require di-catechol, and folates were determined by HPLC and liquid THF as a cofactor. On the addition of THF to cell-free extracts, chromatography (LC)/MS/MS. After 30 min incubation, sesamin the sesamin-metabolizing activity increased 350 times (Table 1 mono- and di-catechol increased to 77 and 9.3 μM, respectively. and SI Appendix, Table S3). Coincidentally, sesamin decreased by 80 μM(SI Appendix,Fig.S11A). SesA was purified as a single band on SDS/PAGE (Table 1 We detected 5,10-CH2-THF in SesA reaction mixtures. After μ and SI Appendix, Fig. S5) after ammonium sulfate precipitation 30-min incubation, 5,10-CH2-THF increased to 85 M(SI (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 75 SesA was calculated to be 50,385 Da, which was consistent with sesamin that of the purified enzyme determined by SDS/PAGE. These 50 results indicate that SesA consists of three identical subunits. B A 25 Identification of the Sesamin-Metabolizing Enzyme. We determined the draft genome sequence for strain no. 22 using a next-generation 0 sequencer, Hiseq2500 (Illumina). From the sequence information, 0 51015 we identified an ORF of 1,359 nucleotides, 21 of which corre- Abs at 290 nm (mAu) sponded to the above N-terminal sequence (SI Appendix, Supple- Retention time (min) mentary Data 2). The deduced amino acid sequence identified a Fig. 2. HPLC analysis of reaction products of sesamin. Chromatogram of the putative folate-binding domain as in the glycine cleavage T-protein reaction mixture after incubation of sesamin with a cell-free extract of strain (GcvT) (pfam01571). no. 22. The products are indicated as A and B.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1605050113 Kumano et al. Downloaded by guest on October 1, 2021 sesamin mono-catechol, di-catechol, and 5,10-CH -THF were Specific 2 Relative formed stoichiometrically with the consumption of sesamin dur- Substrate activity activity (units/mg) ing the enzymic reaction (Fig. 4A).

10 O O O O 3 7’ Kinetic Analysis. Using varying concentrations of sesamin in the H 8 4’ H O 1 1’ O O OH 100% 4 8’ H H 7 3’ presence of 1 mM THF, a typical hyperbolic curve of product O O 10’ O OH sesamin formation over substrate concentration was obtained (SI Appen- 1 – HO O dix,Fig.S11C), indicating that the reaction followed Michaelis H HO OH Menten kinetics. Apparent steady-state kinetic constants were H O OH estimated for sesamin by quantitative measurements of sesamin 2 monocatechol by HPLC analysis. Nonlinear regression analysis O O HO O H H revealed the following: Km = 0.032 ± 0.005 mM, Vmax = 9.3 ± O O HO O 31% − − − H H μ · 1· 1 = ± 1 O O O O 0.4 ( mol min mg ), and kcat 7.9 0.3 s . (+)-episesamin 3 Substrate Specificity. To examine the substrate specificity of SesA, O O O O H H we investigated the compounds listed in Fig. 3. The structures of O O O OH H H 88% O O O OH the products were determined by LC/MS/MS and NMR analyses. (-)-asarinin 4 SesA catalyzed the demethylenation of (+)-episesamin, (−)-asarinin, HO O + 3 H sesaminol, and ( )-sesamolin to yield monocatechols (SI Appendix, HO OH H Figs. S12–S16), 4 (SI Appendix,Figs.S17–S22), 6 (SI Appendix,Figs. O OH – 8 9 – 5 S23 S25), and and (SI Appendix,Figs.S26 S28), and di-catechols O O 5 – 7 H (SI Appendix, Figs. S17 and S29 S32), (SI Appendix,Fig.S23), O O H no products and 10 (SI Appendix,Fig.S26). Moreover, piperine (derived O O diasesamin from black pepper) was also demethylated (SI Appendix,Figs. O O O O S33–S35). SesA activity was specific for the methylenedio- H H O O O OH H H 112% xyphenyl and not the methoxyphenyl group. O O O OH OH OH sesaminol 6

Mutational Analysis of SesA. To investigate the reaction mecha- MICROBIOLOGY HO O H nism of SesA, we constructed a set of mutants with single-amino HO OH H O OH acid substitutions. SesA was found to belong to a diverse family OH 7 of enzymes that include specific domains of dimethylglycine

O O O O (DMGO) (23), oxidase (24), dimethylsulfo- H H O O O OH niopropionate demethylase (DmdA) (25), and demethylase. The O H O H 97% O O O OH enzyme acts on lignin-degradation products such as syringate and sesamolin 8 vanillin [DesA (26, 27) and LigM (28), respectively], as well as HO O H GcvT (29). HO O O H O O Based on the amino acid sequence alignment of these enzymes, 9 we prepared three mutants of SesA: D95A, E189A, and Y221A.

HO O Each of the mutant enzymes was expressed in E. coli and purified H HO OH by the method described in Materials and Methods (SI Appendix, O H O OH Fig. S36). Enzymatic activity was measured using the method as 10 that for wild-type SesA. E189A and Y221A exhibited no activity H3CO O H HO OH at all. The activity of the D95A derivative was 40% compared H no products O OCH3 with that of the wild-type enzyme. pinoresinol Discussion N N In nature, many physiologically active compounds, such as fla- O OH O O 11% vonoids, terpenoids, alkaloids, steroids, coumarins, glycosides, O OH piperine 11 and nucleosides, are produced by plants and microorganisms. Unique pathways of microbial metabolism of these compounds

O O – O O have been reported (30 34). Research on the microbial metab- O H OH O O olism of a diversity of natural compounds can be expected to H O HO OH O reveal novel enzymes (4) and catalytic functions (34). We recently OCH3 OCH3 curcumin* samin* piperonal* reported a curcumin metabolic pathway in E. coli and identified a

HO novel curcumin/dihydrocurcumin reductase (4). Here, we isolated OH H3CO O O O sesamin-catabolizing microorganisms and determined the initial H3CO O HO steps of the sesamin-metabolic pathway at both protein and sesamol* vanillin* isovanillin* gene levels. *no products were observed Sesamin is a major lignan in sesame oil with characteristic methylenedioxyphenyl groups. A methylenedioxyphenyl group Fig. 3. SesA activities for plant-derived methylenedioxyphenyl compounds is present in some plant metabolites, such as berberin (isolated and their derivatives. The specific activities for each substance and structures from Berberis), piperonal (isolated from dill, vanilla, violet flowers, of reaction products are indicated. and black pepper), and piperine (isolated from black pepper), as well as sesamin. Also, drugs such as tadalafil and 3,4-methyl- enedioxymethamphetamine (MDMA) have a methylenedioxyphenyl Appendix,Fig.S11B); there was no change in the 5-CH3-THF group. In humans, the bridges (O-C-O) of amount. THF could not be determined because of its instability methylenedioxyphenyl compounds are oxidized to catechols by under our assay conditions. These results demonstrate that CYP450s (12, 35).

Kumano et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Table 1. Purification of SesA Step Total protein, mg Total activity, units Specific activity, units/mg Yield, % Purification, fold

Cell-free extract 390 120 0.31 100 1

(NH4)2SO4 170 110 0.66 88 2.1 Hiprep Butyl FF 21 30 1.4 25 4.5 Mimetic Orange 1 A6XL 1.2 9.1 7.6 7.5 24

On the other hand, microbial enzymes that metabolize meth- equatorial positions, when the of the tetrahy- ylenedioxyphenyl bridges have not been reported although me- drofuran ring is 8S, 8′S. tabolites have been identified in a few cases. For example, Also, the activity of SesA was affected by substrate size. Pip- sesamin is converted into sesamin monocatechol and sesamin erine was an active substrate whereas small methylene dioxy- di-catechol by A. oryzae (15). In addition, cell-free extracts of phenyl compounds such as samin were inert substrates (Fig. 3). Pseudomonas fluorescens strain PM3 oxidize piperonylic acid into The crystal structure of GcvT of Thermotoga maritima shows protocatechuate and formic acid, requiring NADH or NADPH that aspartic acid (D96), glutamic acid (E195), and tyrosine as a cofactor (36). Recently, it was reported that intestinal bac- (Y100) are hydrogen-bonded to THF (29). These hydrogen bonds teria convert sesamin into mammalian lignans (16). are also observed in the structures of DMGO, , In this study, we identified SesA, which converts sesamin into and DmdA; sequence alignment of SesA, LigM, DesA, GcvT, sesamin mono- and di-catechol. The cleaved methylene group DmdA, and DMGO demonstrated that D95 and E189 of SesA of sesamin is transferred to THF, 5,10-CH2-THF being formed. corresponded to D96 and E195 of GcvT (SI Appendix,Fig.S38). Thus, SesA is a THF-dependent sesamin/sesamin-monocatechol Tyrosine is not conserved at the corresponding position in the methylenetransferase. In this reaction, the methylene group protein sequences of GcvT family proteins. Strictly speaking, Y100 from the methylenedioxy bridge is transferred without an apparent of GcvT, Y660 of DMGO, and Y206 of DmdA are hydrogen- change in state to THF. This mechanism is distinct from that bonded to THF. In this alignment, Y221 of SesA corresponded of CYP450 (12), which oxidatively removes the methylene group to Y247 and Y242 of LigM and DesA, respectively. The E189A of sesamin. Compared with the enzymatic activity of SesA and and Y221A mutants of SesA were inactive. Considering the CYP2C9, which contributes significantly to the metabolism of ses- reported crystal structures of DmdA (25) and GcvT (29), this amin in the liver, the Km values of SesA and CYP2C9 were 32 μM and 5.4 μM, respectively. On the other hand, the kcat value of SesA for sesamin was 220 times higher than that of CYP2C9 (12). O O thf1 sesA thf2 Homology searches of the protein database demonstrated that A H B ∼ O O SesA exhibits low similarity ( 20%) to GcvT, which is involved H O O in the (GCS) together with P-protein, Sesamin H-protein, and L-protein. In general, GCS is found in most bac- Thf1 1 kb THF teria, archaea, and the mitochondria of all eukaryotes and plays SesA 10-CHO-THF critical roles in both glycine degradation and one-carbon metab- 5,10-CH -THF 2 Thf2 olism (37). O O H The folate-binding domain of GcvT (pfam01571) is conserved OH Nucleotides O Folate-dependent in SesA and also in DMGO, sarcosine oxidase, DmdA, DesA, H Vitamins O OH one carbon pool and LigM. These GcvT family proteins exhibit weak sequence Sesamin mono-catechol Amino acids Thf1 identities and are classified into separate clades from one an- THF other (SI Appendix, Fig. S37). SesA 10-CHO-THF SesA is distinct as follows: (i) SesA is a homo-trimer that 5,10-CH -THF 2 Thf2 forms no complex with any other proteins; (ii) the substrates of HO O SesA are aromatic compounds; and (iii) SesA catalyzes a ring H HO OH cleavage reaction to transfer the methylene group to THF (Fig. 1). H O OH In particular, point iii is unique to SesA among the GcvT family Sesamin di-catechol proteins. SesA, LigM, and DesA all metabolize aromatic compounds although the sequence similarities of SesA to LigM and DesA TCA cycle are only 22% and 26%, respectively. On the other hand, SesA showed distinct enzymatic activities compared with LigM and C R' R' R' DesA. LigM and DesA transfer the “methyl” group to THF, “ ” O HO HO R yielding 5-CH -THF. On the contrary, SesA transferred the BH B BH OH 3 O HB' O HB' B' CH O N “ ” “ ” O H O O 2 methylene group of sesamin to THF, giving 5,10-CH2-THF. H N N5 R N R N R HN HN N10 HN N HN N Although we investigated each of the compounds listed in Fig. 3, H H H H N N N H2N N N H2N N N H2N N N 2 we found that SesA, does not transfer the to THF. H H H H The enzymatic activity of SesA was influenced by the stereo- (i) (ii) (iii) (iv) chemistry of the methylenedioxyphenyl group (Fig. 3). Dia- sesamin and (+)-episesamin monocatechol are inert substrates Fig. 4. Sesamin metabolic pathway and proposed reaction mechanism. (Fig. 3), suggesting that SesA catalyzes only the demethylenation (A) Proposed sesamin metabolic pathway in strain no. 22. (B) DNA sequence of an equatorial methylenedioxyphenyl group, when the stereo- of region encoding SesA and its flanking region. The thfl gene exhibits 60% amino acid sequence identity with that of Mycobacterium bovis (UniProtKB chemistry of the tetrahydrofuran ring is 8R, 8′R. On the other − − accession code P0A5T7), and the thf2 gene exhibits 69% amino acid se- hand, ( )-asarinin is converted into ( )-asarinin di-catechol, quence identity with that of Clavibacter michiganensis (UniProtKB accession which suggests that SesA is able to catalyze the demethylenation code B0RD2). (C) Proposed reaction mechanism for SesA. In C, “B” represents of methylenedioxyphenyl groups that are in both axial and a base.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1605050113 Kumano et al. Downloaded by guest on October 1, 2021 finding suggests that E189 and Y221 of SesA would be hydrogen- 5,10-CH2-THF produced by the SesA reaction would be me- bonded to THF. tabolized as follows. Analysis of the gene annotation of strain no. The reaction mechanisms of GcvT, DmdA, and DMGO were 22 indicated that strain no. 22 has a putative formyltetrahydrofolate proposed based on 3D structural and mutational analyses (23, deformylase gene (thf1), and a putative 5,10-CH2-THF 25, 38). (i) In GcvT of E. coli, the electron relay from D97 (or dehydrogenase/cyclohydrolase gene (thf2) upstream and down- D96) to N113 through the hydrogen bond could make N113 act stream of the sesA gene, respectively (Fig. 4B). Thf2 could as a base to deprotonate the protonated amino group of the convert 5,10-CH2-THF into 10-formyltetrahydrofolate (10-CHO- aminomethyllipoyllysine arm in the substrate, followed by the THF), which is a substrate for the biosynthesis of purine. Also, cleavage of the C–S bond of the arm after migration of a proton Thf1 could convert 10-CHO-THF into THF. These findings sug- from the protonated R223 to the substrate, yielding a reactive gest that strain in sesamin no. 22 is important physiologically. iminium intermediate (38). The iminium intermediate reacts Some SesA homologs, which we found by Blast searches, form with the N5 of THF to form and an iminium ion a gene cluster with folate-metabolizing enzyme genes (SI Ap- including the N5 atom of THF. Next, D97 deprotonates the N10 pendix, Fig. S39). Most were derived from Actinobacteria, but atom of 5-CH2-THF, and this deprotonated N10 attacks the this gene set was also observed in the Gram-negative bacterium, iminium ion including N5, to form 5,10-CH2-THF. (ii) In DmdA, Bradyrhizobium japonicum WSM2793. These findings suggest methyl transfer is suggested to be coupled with proton transfer that THF-dependent C1 transferases are distributed in various that is initiated by a base and mediated by a water in microorganisms. the . In this reaction, as a nucleophile, Sp3 hybridized-N5 of THF attacks the CH3 group on the sulfonium ion of the sub- Materials and Methods strate, to yield 3-(methylthio)propionic acid (25). Therefore, the Bacterial Strains, Plasmids, Primers, and Additional Methods. For bacterial proton donor is not required in this reaction. (iii) In DGMO, THF strains, plasmids, and primers, see SI Appendix, Tables S1 and S2. attacks the iminium ion of the substrate via the nucleophilic N10 For chemicals, HPLC and LC/MS/MS analyses, structure determination, atom, with concomitant deprotonation by D552, followed by the purification of the sesamin-metabolizing enzyme from Sinomonas sp. no. 22, formation of sarcosine and 5,10- CH -THF through intramolecular the draft genome sequence of Sinomonas sp. no. 22, cloning and heterol- 2 ogous expression of sesA, determination of the molecular mass of SesA, time rearrangement of the covalent intermediate formed between THF courses of cell growth and enzymatic activity, Western blot analysis, mea- and the iminium intermediate (i.e., N5 of THF attacks on the surement of folate, temperature dependency and stability, pH dependency covalent intermediate with concomitant deprotonation of N5 of and stability, substrate specificity, circular dichroism analysis, and site-directed

THF by the nascent sarcosine) (23). mutagenesis, see SI Appendix, Supplementary Methods. MICROBIOLOGY 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. Isolation of Sesamin-Metabolizing Microorganisms. Sesamin-metabolizing Considering this finding and the proposed reaction mechanisms of microorganisms were isolated from soil in the University of Tsukuba and sesame other GcvT family enzymes, direct nucleophilic attack on sesamin gardens by the following enrichment method. Step 1 was as follows: 1 g of by N5 of THF would initiate the reaction, as seen in the case of collected soil was added to 10 mL of sesamin medium, which consisted of 0.1% DmdA. However, the reaction mechanism of SesA is not the same (wt/vol) sesamin, 1% (wt/vol) (NH4)2SO4, 0.05% (wt/vol) KH2PO4,0.05%(wt/vol) K HPO , 0.05% (wt/vol) MgSO ·7H O, 0.0005% (wt/vol) FeSO ·7H O, and as other members of the GcvT family. In the SesA reaction, the 2 4 4 2 4 2 – – 10% (vol/vol) tap water, adjusted to pH 7.0 with NaOH, followed by incubation methylenedioxy groups (O C O) are cleaved to yield OH groups at 28 °C or 37 °C for 3 d. Step 2 was as follows: 2% (vol/vol) of the cultivated of catechol moieties; proton donation is required to cleave the medium was added to the same fresh medium, followed by incubation at O–C–O bond, which is not present in substrates of other GcvT 28 °C or 37 °C for 3 d. Step 2 was repeated three times. family enzymes. Therefore, we propose a possible reaction After enrichment, the culture broth was spread on sesamin sole-carbon + + mechanism, in which proton donors (indicated by BH and B′H agar plates, which contained 1.5% (wt/vol) agar in addition to the above in Fig. 4C) are involved. In previously reported reaction mech- sesamin sole-carbon medium, and colonies that grew on these plates on 1 wk anisms of GcvT family enzymes, proton donation does not occur incubation at 28 °C were isolated. except in GcvT. In GcvT, R223 is predicted to donate a proton to Each of the isolated strains was inoculated into a test tube containing the substrate for cleavage of the C–S bond. According to the 10 mL of sesamin sole-carbon medium, followed by incubation at 28 °C for 2 d. Cells were harvested by centrifugation (4,000 × g,10min,4°C)and,after amino acid alignments of these proteins, an residue, washing twice with 10 mM potassium phosphate buffer (KPB) (pH 7.0), were which corresponds to R223 in GcvT, is not conserved in SesA, resuspended in 200 μL of the same buffer. Then, the cells were disrupted by LigM, and DesA. We predict that the amino acid residues act as sonication, and the cell debris was removed by centrifugation (27,000 × g, proton donors that provide the methylenedioxy bridges of ses- 10 min, 4 °C) to prepare a cell-free extract. Two hundred microliters of the amin with a proton. Considering the crystal structures of GcvT, reaction mixture comprised 10 μL of 100 mM KPB (pH 7.0), 10 μLof10mM DmdA, and DMGO in the GcvT family proteins, candidates of sesamin (in DMSO), 100 μL of the cell-free extract, and milliQ water. After proton donor residues in the predicted active site of SesA are as incubation at 28 °C for 16 h, the reaction was stopped by adding 100 μLof follows: R81, H82, R100, R179, and H225 (SI Appendix, Fig. acetonitrile. The reaction samples were analyzed by HPLC and LC/MS. + + S38). BH and B′H provide protons and become :B and :B′, respectively, in sesamin. In the proposed reaction (Fig. 4C), the 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, ring closure to yield 5,10-CH2-THF is predicted to be initiated by μ · μ μ ′ ′ 5 L of 1 M Tris HCl (pH 8.0), 3 L of 10 mM substrate (in DMSO), 10 Lof a base (:B ). Then, the :B should accept the proton on the N10 10 mM THF, and 2 μL of Tween 80 were used]. THF was dissolved in 50 mM atom of an iminium ion, including the N5 atom of 5-CH2-THF in Tris·HCl (pH 9.0), 1% 2-mercaptoethanol, and 2% (wt/vol) ascorbate. One the last step (step iii). The SesA reaction is different from that of unit of sesamin-converting activity was defined as the amount of enzyme other GcvT family enzymes in that SesA requires proton donors required to catalyze the formation of 1 μmol of sesamin monocatechol per for the reaction. To identify the amino acid residues B and B′, minute. Specific activity is expressed as units per milligram of protein. studies on the crystal structure and site-directed mutagenesis The reaction was initiated by adding the enzyme, followed by incubation at 28 °C for an appropriate time. After incubation, the reaction was stopped studies of SesA are required. μ At the beginning of this study, we isolated strain no. 22 by by adding 100 L of acetonitrile. For determination of the kinetic parameters for the demethylenation of enrichment culture using sesamin as a sole-carbon source. In the sesamin, 100 μL of the reaction mixture consists of 1 μL of 0.0731 mg/mL growth experiment, the enzymatic activity of SesA was found to SesA, 5 μL of 1 M Tris·HCl (pH 8.0), 10 μLof10mMTHF,2μL of Tween 80, and increase just before the onset of cell growth (SI Appendix,Fig.S7). from 0.05 mM to 0.3 mM sesamin. The reactions were initiated by the ad- These experiments and Western-blot analysis revealed that SesA dition of SesA, followed by incubation at 28 °C, and then termination at 1, 3, formation was induced by sesamin in both media. Moreover, 5, 7, and 10 min by the addition of 50 μL of acetonitrile. The experiments

Kumano et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 were carried out in duplicate independently. The kcat values were calculated numbers LC101493 for sesA and thf2, LC101494 for thf1, and LC101495 for using a Mr of 50,385 for SesA. the 16S rRNA gene.

Nucleotide Sequence Accession Numbers. The nucleotide sequence data ACKNOWLEDGMENTS. We thank Dr. Kentaro Shiraki (University of Tsukuba) reported in this paper appear in the DDBJ/GenBank database under accession for help with circular dichroism spectra analysis.

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