plants

Article Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes

Mariko Baba 1 , Ken-ichi Yamada 2 and Michiho Ito 1,*

1 Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan; [email protected] 2 Department of Pharmaceutical Organic Chemistry, Graduate School of Pharmaceutical Sciences, Tokushima University, Shomachi, Tokushima 770-8505, Japan; [email protected] * Correspondence: [email protected]



Received: 14 April 2020; Accepted: 28 April 2020; Published: 1 May 2020


Abstract: Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.

Keywords: phenylpropanoid; volatile compound; molecular cloning; cytochrome P450; biosynthetic pathway; perilla

1. Introduction Perilla (Perilla frutescens Britton var. crispa W. Deane) is a common culinary herb in East and Southeast Asia, and types of perilla that principally contain perillaldehyde, a monoterpene (MT) compound, in their essential oils are used pharmaceutically in China and Japan. Essential oils of perilla can be classified into more than 10 types [1], which are roughly divided into two groups based on the structures of their constituents [2]: MT-type oils, and phenylpropene (PP)-type oils whose main constituents are elemicin, myristicin, dillapiole, and nothoapiole (Figure1). This biosynthetic pathway of perilla oil is genetically controlled [3,4], and the functions of each gene can be determined by cloning the enzymes that catalyze the relevant reaction steps in the biosynthetic pathway. The enzymes that catalyze the formation of MT-type oils, such as limonene synthase, geraniol synthase, linalool synthase, and perillaldehyde synthase, were previously characterized [5–7], whereas no PP-type synthases have been reported to date. Known phenylpropanoid volatile components include anethole in fennel, apiole in parsley, eugenol in clove, and myristicin in nutmeg (Figures1 and2), and their pharmacological actions and toxicities are well studied due to their use as pharmaceuticals and as

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flavorings.(Figures For 1 and example, 2), and myristicintheir pharmacological is used to actions treat rheumatism and toxicities and are anxietywell studied in traditional due to their medicine. use Risk assessmentas pharmaceuticals of myristicin and as usingflavorings. the margin For example, of exposure myristicin approach is used has to been treat conductedrheumatism because and of anxiety in traditional medicine. Risk assessment of myristicin using the margin of exposure its potentialanxiety genotoxicityin traditional ormedicine. carcinogenicity Risk assessment [8]. Dillapiole, of myristicin which using is found the margin mainly of in dill,exposure has been approach has been conducted because of its potential genotoxicity or carcinogenicity [8]. Dillapiole, reported to have cytotoxic effects [9]. However, only a few enzymes involved in the synthesis of these which is found mainly in dill, has been reported to have cytotoxic effects [9]. However, only a few compounds have been isolated and reported, and these known enzymes catalyze the formation of enzymes involved in the synthesis of these compounds have been isolated and reported, and these compoundsknown enzymes with simple catalyze side the groups formation [10]. of compounds with simple side groups [10].

FigureFigure 1. Putative 1. Putative biosynthetic biosynthetic pathway pathway of of phenylpropenephenylpropene (PP)-type (PP)-type oils oils in inperilla. perilla. In the In text, the text,the oil the oil typestypes of each of straineach strain of perilla of perilla are represented are represente as PP-em,d as PP-em, PP-m, PP-m, PP-md, PP-md, PP-emd, PP-emd, and PP-mdn,and PP-mdn, meaning that eachmeaning strain that mainly each strain contains mainly elemicin contains+ myristicin,elemicin + myristicin, myristicin, myristicin, myristicin myristicin+ dillapiole, + dillapiole, elemicin + myristicinelemicin+ dillapiole,+ myristicin and + dillapiole, myristicin and+ myristicindillapiole + +dillapiolenothoapiole, + nothoapiole, respectively. respectively.

FigureFigure 2. Chemical 2. Chemical structures structures ofof severalseveral phenylpropanoid volatile volatile compounds. compounds.

PerillaPerilla plants, plants, whose whose major major aromatic aromatic compoundcompound is is perillaldehyde, perillaldehyde, are are used used as herbal as herbal medicine medicine for Kampofor Kampo prescriptions prescriptions in in Japan. Japan. Those Those perilla perilla plants that that are are rich rich in inperillaldehyde perillaldehyde do not do contain not contain (E)-asarone (Figure 2) [11]. (E)-Asarone is structurally similar to PP-type oil components [12,13] and (E)-asarone(E)-asarone (Figure (Figure2)[ 112) ].[11]. (E)-Asarone (E)-Asarone is is structurally structurally similar similar to PP-type oil oil components components [12,13] [12,13 and] and is is likely formed through a PP-type oil component pathway, although the details are currently likelyis formed likely formed through through a PP-type a PP-type oil component oil componen pathway,t pathway, although although the details the details are currently are currently unknown. unknown. However, the (Z)-asarone isomer was reported to be toxic [14] and (E)-asarone may be However, the (Z)-asarone isomer was reported to be toxic [14] and (E)-asarone may be carcinogenic [15], carcinogenic [15], and therefore it should always be certified that no perilla plants containing and therefore(E)-asarone it shouldare present always in the be plant certified material that noused perilla to prepare plants the containing pharmaceutical (E)-asarone formulation. are present In in the plantorder material to find usedplants to including prepare thethis pharmaceuticaltoxic compound formulation.and remove them In order from to medicine find plants ingredients, including this toxicpurity compound tests using and remove liquid chromatography, them from medicine optimized ingredients, to separate purity perillaldehyde tests using and liquid (E)-asarone, chromatography, are optimizeddefined to in separate the Japanese perillaldehyde Pharmacopoeia. and (E)-asarone, are defined in the Japanese Pharmacopoeia. The mainThe main constituents constituents of of PP-type PP-type perilla perilla oiloil are elemicin, elemicin, myristicin, myristicin, dillapiole, dillapiole, and andnothoapiole, nothoapiole, and areand likely are likely synthesized synthesized from from phenylalanine phenylalanine (Figure(Figure 11).). The The reaction reaction steps steps to toall allfour four compounds compounds are genetically controlled based on crossing experiments using pure strains developed by repeated are geneticallyare genetically controlled controlled based based on on crossing crossing experimentsexperiments using using pure pure strains strains developed developed by repeated by repeated self-pollination, and on detailed gas chromatography (GC) analyses of perilla essential oils [2,16]. self-pollination,self-pollination, and and on on detailed detailed gas gas chromatography chromatography (GC) (GC) analyses analyses of ofperilla perilla essential essential oils [2,16]. oils [ 2,16]. Previous reports on the synthesis of alkaloids suggested that methoxy or methylenedioxy groups are Previous reports on the synthesis of alkaloids suggested that methoxy or methylenedioxy groups are formed in the synthetic pathways leading to PP-type oil components and may involve hydroxylation formedor cyclization in the synthetic reactions pathways by cytochrome leading P450 to PP-type(hereafter, oil “P450”) components [17]. P450 and enzymes may involve catalyze hydroxylation different or cyclizationreactions, reactionssuch as oxidation, by cytochrome hydroxylation, P450 (hereafter, epoxidation, “P450”) and dealkylation, [17]. P450 enzymesand are involved catalyze in dithefferent reactions,synthesis such of as many oxidation, plant secondary hydroxylation, metabolites. epoxidation, Their low and substrate dealkylation, specificity and and are high involved reaction in the synthesisregio- ofand many stereo-specificity plant secondary are beneficial metabolites. for generating Their lowuseful substrate compounds, specificity making andtheir high catalytic reaction regio-mechanisms and stereo-specificity an attractive target are beneficial of study. for generating useful compounds, making their catalytic mechanismsHere an we attractive describe targetthe isolation of study. of a P450 enzyme involved in the synthesis of dillapiole in PP-type perilla oils using expressed sequence tag (EST) libraries of pure strains of perilla. The perilla HerePP-type we perilla describe oils theusing isolation expressed of asequence P450 enzyme tag (EST involved) libraries in of the pure synthesis strains of of perilla. dillapiole The perilla in PP-type strains have been developed and maintained for more than 30 years through repeated perilla oils using expressed sequence tag (EST) libraries of pure strains of perilla. The perilla strains have been developed and maintained for more than 30 years through repeated self-pollination. The high purity of these strains allows for the selection of P450 sequences that may be involved in the biosynthesis of each oil component by comparing the expression levels of those sequences [18]. Plants 2020, 9, x FOR PEER REVIEW 3 of 19

self-pollination. The high purity of these strains allows for the selection of P450 sequences that may be involved in the biosynthesis of each oil component by comparing the expression levels of those

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2. Results 2. Results 2.1. Isolation of a P450 Sequence from Perilla 2.1. Isolation of a P450 Sequence from Perilla P450-like sequences that are specifically expressed in perilla strains whose essential oils contain mainlyP450-like dillapiole sequences were identified. that are specifically Contig 49487 expressed was selected in perilla from strains our EST whose library essential because oils it containhad the mainlylargest difference dillapiole werein reads identified. per kilobase Contig of 49487 exon wasper selectedmillion mapped from our read EST (RPKM) library because values itof hadthe thesequences largest studied difference (Table in reads 1). We per PCR-amplified kilobase of exon the selected per million contig mapped using readprimers (RPKM) based values on sequence of the sequencesinformation studied from (Tablethe EST1). Wedatabase, PCR-amplified using comp the selectedlementary contig DNA using (cDNA) primers as based a template, on sequence then informationconfirmed the from size the of EST the database,PCR product using by complementary agarose-gel electrophoresis. DNA (cDNA) asThe a template,size of contig then 49487 confirmed was thein accordance size of the PCR with product the sequence by agarose-gel information. electrophoresis. The full-length The size ofsequence contig 49487 of contig was in accordance49487 was withdetermined the sequence by RACE information. methods. The sequence full-length Pf-49487 sequence (GenBank of contig Accession 49487 was No. determined LC476554) by encoded RACE methods.505 amino Theacids sequence (57 kD, FigurePf-49487 3), and(GenBank regions Accession characteristic No. of LC476554) P450s, such encoded as a proline-rich 505 amino region, acids (57an kD,oxygen-binding Figure3), and pocket, regions characteristicand a heme-binding of P450s, suchregion, as awere proline-rich conserved region, [19]. an BLAST oxygen-binding analysis pocket,showed andthat a the heme-binding amino acid region,sequence were of conservedPf-49487 shared [19]. BLAST56% identity analysis with showed that of that the the Sesamum amino acidindicum sequence premnaspirodiene of Pf-49487 shared oxygenase-like 56% identity enzyme with (p thatredicted) of the Sesamum(GenBank indicum Accessionpremnaspirodiene No. JP653840) oxygenase-likeand this was the enzyme highest similarity (predicted) among (GenBank all entrie Accessions in the No.database. JP653840) Premnaspirodiene and this was oxygenase the highest is similaritya hydroxylase among classified all entries as in a the member database. of Premnaspirodienethe CYP71D family. oxygenase Pf-49487 is a had hydroxylase a 49% classifiedamino acid as asequence member identity of the CYP71D to the characterized family. Pf-49487 Hyoscyamushad a 49% muticus amino acid premnaspirodiene sequence identity oxygenase to the characterized (GenBank HyoscyamusAccession No. muticus EF569601)premnaspirodiene [20]. Cinnamate oxygenase 4-hydroxylase (GenBank Accession from Arabidopsis No. EF569601) thaliana [20]. Cinnamate(GenBank 4-hydroxylaseAccession No. fromNP180607)Arabidopsis was thalianaa member(GenBank of the AccessionP450 family No. that NP180607) hydroxylates was a the member aromatic of the ring P450 of familyphenylpropanoid that hydroxylates compounds the aromatic and shared ring of 28% phenylpropanoid sequence identity compounds with Pf-49487 and shared at the 28% amino sequence acid identitylevel. Based with onPf-49487 the standardizedat the amino P450 acid nomenclature level. Based on system, the standardized Pf-49487, is P450 in the nomenclature CYP71D subfamily system, Pf-49487and was, named is in the CYP71D558. CYP71D subfamily and was named CYP71D558.

Figure 3.3. AlignmentAlignment of of the amino acidacid sequencessequences ofof Pf-49487Pf-49487.. HPO: Hyoscyamus muticusmuticus premnaspirodiene oxygenase (GenBank(GenBank AccessionAccession No.No. EF569601), C4H: cinnamatecinnamate 4-hydroxylase4-hydroxylase (Arabidopsis(Arabidopsis thaliana)thaliana)(GenBank (GenBank Accession Accession No. No. NP180607). NP180607). The The following following conserved conserved regions regions of P450 of P450 are underlined:are underlined: the proline-richthe proline-rich region, region, the oxygen-bindingthe oxygen-binding pocket, pocket, and theand heme-binding the heme-binding region region from upstream of the sequence. Black background indicates 100% amino acid identity among the three clones and gray background indicates greater than 50% amino acid identity. Plants 2020, 9, x FOR PEER REVIEW 4 of 19 Plants 2020, 9, x FOR PEER REVIEW 4 of 19

from upstream of the sequence. Black background indicates 100% amino acid identity among the from upstream of the sequence. Black background indicates 100% amino acid identity among the Plantsthree2020, 9clones, 577 and gray background indicates greater than 50% amino acid identity. 4 of 18 three clones and gray background indicates greater than 50% amino acid identity.

Table 1. Reads per kilobase of exon per million mapped read (RPKM) values of contig 49487 and Table 1.1. Reads per kilobase of exon per million mappedmapped read (RPKM) values of contig 49487 and 77404. Oil type C means that the strain mainly contains citral, a monoterpene (MT)-type oil 77404. OilOil typetype C meansC means that that the strainthe strain mainly mainly contains contains citral, a monoterpenecitral, a monoterpene (MT)-type oil(MT)-type component. oil component. Contig 49487 had the largest difference in RPKM values between PP-type strains component.Contig 49487 Contig had the 49487 largest had diff erencethe largest in RPKM differe valuesnce betweenin RPKM PP-type values strains between containing PP-type dillapiole strains containing dillapiole (i.e., strains 16, 25, and 5316) and others. Contig 77404 showed the second (i.e.,containing strains dillapiole 16, 25, and (i.e., 5316) strains and 16, others. 25, and Contig 5316) 77404and others. showed Contig the second 77404 largestshowed di thefference second in largest difference in RPKM values. largestRPKM values.difference in RPKM values. Strain (Oil Type) Strain (Oil Type) Contig 10 (PP-em) 12 (PP-m) 16 (PP-md)Strain (Oil25 (PP-emd) Type) 5316 (PP-mdn) 5717 (C) Contig 10 (PP-em) 12 (PP-m) 16 (PP-md) 25 (PP-emd) 5316 (PP-mdn) 5717 (C) Contig49487 10 (PP-em)0.0824 12 (PP-m) 0.0968 16 (PP-md) 68.4 25 23.7 (PP-emd) 5316 56.7 (PP-mdn) 0.325 5717 (C) 49487 0.0824 0.0968 68.4 23.7 56.7 0.325 77404 0.260 0.459 1.10 1.40 1.70 0.446 4948777404 0.0824 0.260 0.0968 0.459 68.4 1.10 1.40 23.7 1.70 56.7 0.446 0.325 77404 0.260 0.459 1.10 1.40 1.70 0.446 2.2. Comparison of Pf-49487 Expression among Strains of Phenylpropene (PP)-Type Perilla 2.2. Comparison of Pf-49487 Expression among Strains of Phenylpropene (PP)-Type Perilla Quantitative reverse transcription-PCR (RT-PCR) was used to compare the Pf-49487 expression 2.2. ComparisonQuantitative of Pf-49487reverse transcription-PCR Expression among Strains(RT-PCR) of Phenylpropene was used to compare (PP)-Type the Perilla Pf-49487 expression levels in various strains using RNAs isolated from different oil types of perilla: strain 12 of PP-m, levelsQuantitative in various strains reverse using transcription-PCR RNAs isolated (RT-PCR) from different was used oil totypes compare of perilla: the Pf-49487 strain 12expression of PP-m, strain 25 of PP-emd, and strain 5316 of PP-mdn (Figures 1 and 4). The difference in expression level strainlevels in25 variousof PP-emd, strains and using strain RNAs 5316 isolated of PP-mdn from (Figures different 1 oiland types 4). The of perilla: difference strain in 12 expression of PP-m, strainlevel among oil types is similar to the RPKM values in the EST library used for the isolation of Pf-49487, among25 of PP-emd, oil types and is strainsimilar 5316 to the of PP-mdnRPKM values (Figures in1 the and EST4). Thelibrary di ffusederence for in the expression isolation levelof Pf-49487 among, namely, strain 12: 0.0968, strain 25: 23.7, strain 5316: 56.7 (Table 1). These ratios are believed to be namely,oil types strain is similar 12: 0.0968, to the RPKM strain values25: 23.7, in strain the EST 5316: library 56.7 used(Table for 1). the These isolation ratios of arePf-49487 believed, namely, to be relevant to the dillapiole content (%) in oils, namely, strain 12: 0%, strain 25: 9.01%, strain 5316: relevantstrain 12: to 0.0968, the dillapiole strain 25: content 23.7, strain (%) 5316:in oils, 56.7 namely, (Table1 strain). These 12: ratios 0%, strain are believed 25: 9.01%, to be strain relevant 5316: to 21.13% [4,16]. 21.13%the dillapiole [4,16]. content (%) in oils, namely, strain 12: 0%, strain 25: 9.01%, strain 5316: 21.13% [4,16].

Figure 4. The relative expression levels of Pf-49487 in strains 12, 25, and 5316. Error bars indicate SE Figure 4. The relative expressionexpression levelslevels of ofPf-49487 Pf-49487in in strains strains 12, 12, 25, 25, and and 5316. 5316. Error Error bars bars indicate indicate SE SE of of triplicate analyses of two independent samples. oftriplicate triplicate analyses analyses of twoof two independent independent samples. samples. 2.3. Heterologous Expr Expressionession and Functional Analysis of Pf-49487 2.3. Heterologous Expression and Functional Analysis of Pf-49487 P450 enzymes with a bound carbon monoxide (CO) molecule show an absorptionabsorption spectrum P450 enzymes with a bound carbon monoxide (CO) molecule show an absorption spectrum peak at ca. 450 nm, and heterologously expressed P450 enzymes with such an absorption maximum peak at ca. 450 nm, and heterologously expressed P450 enzymes with such an absorption maximum generally have high activity. The The reduced CO-difference CO-difference spectrum of the microsomal fraction of generally have high activity. The reduced CO-difference spectrum of the microsomal fraction of yeast harboring the Pf-49487 expression plasmid was measured and a peak at 448 nm was observed, yeast harboring the Pf-49487 expression plasmid was measured and a peak at 448 nm was observed, suggesting that Pf-49487 has P450 activity (Figure5 5).). suggesting that Pf-49487 has P450 activity (Figure 5).

Figure 5. The reduced carbon monoxide (CO)-difference spectrum of the microsomal fraction of yeast

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(CO)-differencespectrometry plasmid.(MS). spectrum The(MS). of thereaction Themicrosomal reactionproduct fraction ofproduct was was Figurea 5. TheyeastTheFiguresubstrate reduced harboringcatalytic 5. The carbon reactionbyreducedthe Pf-49487 monoxideGC-mass carbonof Pf-49487 expression (CO)-differencemonoxide spectrometry was plasmid. analyzed(CO)-difference spectrum using(MS). of thespectrum the microsomal microsomalThe of thereaction microsomalfractionfraction ofandproduct fraction myristicin of was as a6-allyl-4-methoxy-1,3-benzodioxol-5-ol yeastsubstrate6-allyl-4-methoxy-1,3-benzodioxol-5-ol harboring Figureby the 5.Pf-49487 GC-massThe reduced expression carbonspectrometry (Table plasmid. monoxide 2, (Tablecompound (CO)-difference(MS). 2, compound 20),The a hydroxylated spectrum 20),reaction a hydroxylated of themyristicin productmicrosomal myristicin (Figure wasfraction (Figure of TheFigureyeast catalytic harboring6-allyl-4-methoxy-1,3-benzodioxol-5-ola 5. Theyeastsubstrate reactionreduced the harboring Pf-49487 carbonof byPf-49487 the expression Pf-49487monoxideGC-mass was plasmid. expression analyzed(CO)-difference spectrometry (Table plasmid. using 2, thespectrum compound microsomal(MS). of the 20), microsomalfractionThe a hydroxylated andreaction fraction myristicin myristicin ofproduct as (Figurewas 6-allyl-4-methoxy-1,3-benzodioxol-5-ol6). WhenThe catalytic6). methyl WhenTheyeast reaction catalytic eugenol harboringmethyl of reaction Pf-49487oreugenol the elemicinPf-49487 of was orPf-49487(Table expression elemicinanalyzedwere 2, was compoundus plasmid.ed usingwereanalyzed as theussubstrates, 20),ed microsomalusing asa hydroxylated thesubstrates, the microsomal fraction hydroxylated themyristicin and fractionhydroxylated myristicin products (Figureand myristicinas products as a yeastsubstrate harboring6).6-allyl-4-methoxy-1,3-benzodioxol-5-ol WhenTheby catalyticthe methyl Pf-49487GC-mass reaction eugenolexpression ofspectrometry Pf-49487or plasmid. elemicin was (Table analyzedwere(MS). 2, compoundus usingedThe as the substrates, 20),microsomalreaction a hydroxylated the productfraction hydroxylated myristicinandwas myristicin products (Figure as 6).6-allyl-2,3-dimethoxyphenola WhenThesubstrate catalytic6-allyl-2,3-dimethoxyphenol a methyl substrate reactionby eugenol GC-mass of by Pf-49487or(Table elemicin GC-mass 2,spectrometry was (Tablecompound analyzedwere 2,spectrometry compoundus 5)ed using and(MS).as thesubstrates,6-allyl-2,3,4-trimethoxyphenol 5) microsomal andThe(MS). 6-allyl-2,3,4-trimethoxyphenol thereaction The fractionhydroxylated reaction andproduct myristicin (Tableproducts productwas as2, (Table was 2, 6-allyl-4-methoxy-1,3-benzodioxol-5-olThe catalytica6-allyl-2,3-dimethoxyphenol6). WhensubstrateThe reaction catalytic methyl ofby reaction Pf-49487 eugenolGC-mass of was (TablePf-49487(Tableor analyzed elemicin 2,2,spectrometry was compoundcompound using analyzedwere the us 20), microsomal5) edusing (MS). andaas hydroxylated thesubstrates, 6-allyl-2,3,4-trimethoxyphenol microsomal Thefraction myristicinreactionthe and fraction hydroxylatedmyristicin (Figure andproduct asmyristicin (Tableproducts was as2, Plants6-allyl-2,3-dimethoxyphenola substrate2020,compound6-allyl-4-methoxy-1,3-benzodioxol-5-ol9, 577 Theby catalytic 31) GC-masswere reaction(Table obtained, 2, ofspectrometry compoundPf-49487 respectively was (Table 5) analyzed (Figureand(MS). 2, 6-allyl-2,3,4-trimethoxyphenolcompound 6). usingThe Dillapiole, the 20),reaction microsomal a eugenol, hydroxylated product fractionlimonene, (Table myristicin and wasand myristicin2, geraniol (Figure5 of as 18 compound6-allyl-4-methoxy-1,3-benzodioxol-5-olThe catalyticcompounda 31) weresubstrate reaction obtained, 31) wereof byPf-49487 respectivelyobtained,GC-mass was (Table respectivelyanalyzed (Figure 2,spectrometry compound using6). (FigureDillapiole, the 20), microsomal 6). a (MS). Dillapiole, eugenol,hydroxylated fractionThe limonene, eugenol, myristicin andreaction limonene,andmyristicin geraniol (Figureproduct andas geraniol was compound6).a6-allyl-4-methoxy-1,3-benzodioxol-5-ol Whensubstrate6-allyl-4-methoxy-1,3-benzodioxol-5-ol 6-allyl-2,3-dimethoxyphenol methyl31) wereby eugenol obtained,GC-mass or respectively elemicin spectrometry (Table(Table were (Figure 2,2, uscompound(Tablecompound ed6). (MS).asDillapiole, 2, compoundsubstrates, 20), 5) The anda eugenol,hydroxylated 6-allyl-2,3,4-trimethoxyphenol20),thereaction a hydroxylatedlimonene, hydroxylated myristicinproduct and productsmyristicingeraniol (Figurewas (Table (Figure 2, awere6). When substratenot substrateswere 6).6-allyl-4-methoxy-1,3-benzodioxol-5-olamethyl When notsubstrateby substrates foreugenol methyl thisGC-mass enzyme by orforeugenol thiselemicin GC-mass (Table enzymespectrometry or 3).wereelemicin (Table spectrometryus (Table ed3).were (MS). as 2, substrates,uscompound edThe (MS).as substrates, the20),reaction The hydroxylateda hydroxylated thereactionproduct hydroxylated products myristicinproductwas products (Figurewas 6-allyl-2,3-dimethoxyphenolwere6-allyl-4-methoxy-1,3-benzodioxol-5-ol not substrates6).werecompound When not forsubstratesmethyl 31) this were enzyme (Tableeugenol for obtained, this (Table2, enzyme orcompound(Table respectively 3).elemicin (Table2, compound 5)were 3). and(Figure us6-allyl-2,3,4-trimethoxyphenol 20),ed 6). aas Dillapiole, hydroxylated substrates, eugenol, themyristicin hydroxylated limonene, (Table (Figure and2, products geraniol 6-allyl-4-methoxy-1,3-benzodioxol-5-ol6-allyl-2,3-dimethoxyphenol6). When 6-allyl-2,3-dimethoxyphenol6-allyl-4-methoxy-1,3-benzodioxol-5-olmethyl eugenol (Tableor elemicin 2, compound(Table were 2,2, compoundus compound(Table 5)ed andas 2, substrates,6-allyl-2,3,4-trimethoxyphenolcompound 20),5) anda hydroxylated 6-allyl-2,3,4-trimethoxyphenol the20), hydroxylateda hydroxylated myristicin (Tableproducts myristicin(Figure 2, (Table (Figure 2, compound6). When6-allyl-2,3-dimethoxyphenol were6). methyl31) When were not eugenolsubstrates obtained,methyl or eugenolforrespectively elemicin this enzyme (Tableor were elemicin(Figure 2,(Table compoundus ed 6). 3).were Dillapiole,as substrates,us 5)ed and aseugenol, 6-allyl-2,3,4-trimethoxyphenol substrates,the hydroxylatedlimonene, the hydroxylatedand productsgeraniol (Tableproducts 2, 6-allyl-2,3-dimethoxyphenolThe catalyticcompound6). When reaction 31)methyl wereTable (Table ofeugenol obtained, 2.Pf-49487 SynthesizedTable 2, compoundor 2.respectively wasSynthesizedelemicin allylphenols analyzed 5) were(Figure andallylphenols used using6-allyl-2,3,4-trimethoxyphenolus ined6). this Dillapiole, as theused study. substrates, microsomalin this eugenol,study. the fraction limonene,hydroxylated (Table and and 2, myristicin productsgeraniol were6).6-allyl-2,3-dimethoxyphenolcompound When not substratescompound 6-allyl-2,3-dimethoxyphenol methyl31) were eugenolfor obtained,31) this were enzyme or(Table obtained, respectivelyelemicin (Table2,Table compound(Table respectively 2.3).were (FigureSynthesized 2, uscompound 5) ed6). (Figureand asDillapiole,allylphenols substrates,6-allyl-2,3,4-trimethoxyphenol 6).5) andDillapiole, eugenol, used 6-allyl-2,3,4-trimethoxyphenolthe in this hydroxylatedlimonene,eugenol, study. limonene, and (Tableproductsgeraniol and 2, geraniol(Table 2, ascompound a substratewere6-allyl-2,3-dimethoxyphenol 31) notwere by substrates GC-massobtained,Table for respectively2. spectrometry thisSynthesized enzyme (Table (Figure allylphenols (Table 2, (MS). compound 6).3). TheDillapiole, used reaction in5) this and eugenol, study. 6-allyl-2,3,4-trimethoxyphenol product limonene, was 6-allyl-4-methoxy-1,3-and geraniol (Table 2, 6-allyl-2,3-dimethoxyphenolcompoundwere not substrateswerecompound 31) notwere substrates for obtained, 31) this were enzyme (Tablefor respectivelyobtained, this (Table2, Tableenzyme compound respectively 2.3). (FigureSynthesized (Table 5) 6).3). and (Figure Dillapiole, allylphenols 6-allyl-2,3,4-trimethoxyphenol 6). Dillapiole,eugenol, used in this limonene, eugenol, study. limonene,and (Table Retentgeraniol and2, Retent geraniol benzodioxol-5-olwere not substratescompound (Table for 31) this2 were, enzyme compound obtained, (Table 20), respectively 3). a hydroxylated (Figure myristicin6). Dillapiole, (Figure eugenol,6). When limonene, methyl and eugenol Retentgeraniol compoundwere not substrateswere 31) were not forsubstrates Nameobtained, thisTable (CompoundenzymeName forrespectively 2. Synthesizedthis (Table (Compound enzyme 3). (Figure allylphenols (Table 6). 3). Dillapiole, used in this eugenol, study. limonene, and Retentgeraniolion ion or elemicinFormulawere were Formula not used substrates asTable substrates, forName 2. thisSynthesizedTable (Compoundenzyme the 2. hydroxylatedSynthesized allylphenols (Table 3). allylphenols used productsMass in this Spectra used study.Mass 6-allyl-2,3-dimethoxyphenol in this Spectra study. (TableRetention 2, were not substratesFormula forName this Number) (Compoundenzyme Number) (TableTable 2. 3). Synthesized allylphenols usedMass in this Spectra study. Timeion Time compoundFormula 5) and 6-allyl-2,3,4-trimethoxyphenolTableName 2. SynthesizedNumber) (Compound allylphenols (Table usedMass2 in, compound this Spectra study. 31) were obtained,Retent respectivelyTimeion Formula Number)Table 2. Synthesized Table 2. Synthesized allylphenols allylphenols used in this usedstudy.Mass in this Spectra study. Retent(min)Time (min)Retent NameTable (Compound 2. SynthesizedNumber)Table 2. Synthesized allylphenols allylphenols used in this usedstudy. in this study. ion Retent(min)Time (FigureFormula6). Dillapiole, Name eugenol, (CompoundName limonene, (Compound and geraniol wereMass Spectra not substrates for this enzymeRetent(min)ion (Tableion3 ). Formula Formula Number)Name (Compound Mass SpectraMass Spectra RetentTime Retent(min)ion FormulaName (CompoundNumber) Mass Spectra ion RetentTime Formula 6-Allyl-2,3-dimethoxyNumber)6-Allyl-2,3-dimethoxyName (Compound Mass Spectra Retent(min)Time ion FormulaName Number) (CompoundTable6-Allyl-2,3-dimethoxy Number) 2. Synthesized allylphenols used inMass this study. Spectra Timeion26 Time26 Formula 6-Allyl-2,3-dimethoxyNamephenol (CompoundName (5)phenol Number) (Compound (5) Mass Spectra (min)ion (min)Timeion26 Formula Formula Number)6-Allyl-2,3-dimethoxy phenol (5) Mass SpectraMass Spectra Time26 (min) phenol (5) Number) (min) Time Number) (min)Time Retention(min)26 Formula 6-Allyl-2,3-dimethoxy Name (Compoundphenol Number) (5) Mass Spectra (min) 6-Allyl-2,3-dimethoxy6-Allyl-2,3-dimethoxy (min)26 Time (min) phenol 6-Allyl-2,3-dimethoxy (5) 26 26 6-Allyl-2,3-dimethoxyphenol (5)phenol (5) 26 2-Allyl-4,5-dimethoxy6-Allyl-2,3-dimethoxy2-Allyl-4,5-dimethoxy6-Allyl-2,3-dimethoxyphenol (5) 26 phenol2-Allyl-4,5-dimethoxy6-Allyl-2,3-dimethoxy (5) 5026 5026 2-Allyl-4,5-dimethoxy6-Allyl-2,3-dimethoxyphenolphenol (8) (5) 50 6-Allyl-2,3-dimethoxyphenol phenol (8)(5) phenol (8) (5) 5026 2626 phenol 2-Allyl-4,5-dimethoxy (8)(5) phenol (5) 50 2-Allyl-4,5-dimethoxy phenol (8) 2-Allyl-4,5-dimethoxy2-Allyl-4,5-dimethoxy 50 phenol 2-Allyl-4,5-dimethoxy (8) 50 2-Allyl-4,5-dimethoxyphenol (8) 50 phenol2-Allyl-4,5-dimethoxy (8) 50 50 5-Allyl-2,3-dimethoxy2-Allyl-4,5-dimethoxy5-Allyl-2,3-dimethoxyphenol (8) 50 2-Allyl-4,5-dimethoxy2-Allyl-4,5-dimethoxyphenolphenol 5-Allyl-2,3-dimethoxy2-Allyl-4,5-dimethoxy (8) (8) 2950 5029 5-Allyl-2,3-dimethoxyphenol (8)phenol phenol (14) (8) 2950 phenol (14)phenol (8) 5029 phenolphenol 5-Allyl-2,3-dimethoxy (14)(8)phenol (14) 29 5-Allyl-2,3-dimethoxy phenol (14) 5-Allyl-2,3-dimethoxy5-Allyl-2,3-dimethoxy 29 5-Allyl-2,3-dimethoxyphenol 5-Allyl-2,3-dimethoxy (14) 29 29 phenol (14)phenol (14) 29 6-Allyl-4-methoxy-1,3-5-Allyl-2,3-dimethoxy5-Allyl-2,3-dimethoxyphenol6-Allyl-4-methoxy-1,3-5-Allyl-2,3-dimethoxyphenol (14) (14) 29 29 6-Allyl-4-methoxy-1,3-phenol6-Allyl-4-methoxy-1,3-5-Allyl-2,3-dimethoxy (14) 29 3729 5-Allyl-2,3-dimethoxy phenol (14) 37 37 benzodioxol-5-olphenol benzodioxol-5-ol6-Allyl-4-methoxy-1,3- (14) (20) (20) 2937 29 phenolbenzodioxol-5-ol (14)phenol (14) (20) 6-Allyl-4-methoxy-1,3-benzodioxol-5-ol (20) 37 6-Allyl-4-methoxy-1,3- 6-Allyl-4-methoxy-1,3-benzodioxol-5-ol (20) 6-Allyl-4-methoxy-1,3-6-Allyl-4-methoxy-1,3- 37 6-Allyl-4-methoxy-1,3-benzodioxol-5-ol (20) 37 3737 6-Allyl-4-methoxy-1,3-benzodioxol-5-olbenzodioxol-5-ol6-Allyl-4-methoxy-1,3-benzodioxol-5-ol (20) (20) (20) 37 37 5-allyl-7-methoxy-1,3-benzodioxol-5-ol6-Allyl-4-methoxy-1,3- (20) 37 6-Allyl-4-methoxy-1,3-5-allyl-7-methoxy-1,3-benzodioxol-5-ol5-allyl-7-methoxy-1,3- (20) 37 5-allyl-7-methoxy-1,3-benzodioxol-5-ol benzodioxol-5-ol (20) (20) 3766 6637 benzodioxol-4-olbenzodioxol-5-ol (23) (20) 66 benzodioxol-5-olbenzodioxol-4-ol5-allyl-7-methoxy-1,3-benzodioxol-4-ol (20)(23) (23) 66 benzodioxol-4-ol 5-allyl-7-methoxy-1,3- (23) 66 5-allyl-7-methoxy-1,3- 66 5-allyl-7-methoxy-1,3- benzodioxol-4-ol5-allyl-7-methoxy-1,3-benzodioxol-4-ol (23) (23) 66 5-allyl-7-methoxy-1,3- 66 5-allyl-7-methoxy-1,3-benzodioxol-4-ol (23) 66 5-allyl-7-methoxy-1,3-benzodioxol-4-ol benzodioxol-4-ol5-allyl-7-methoxy-1,3- (23) (23) 66 66 5-Allyl-6,7-dimethoxy5-Allyl-6,7-dimethoxybenzodioxol-4-ol5-allyl-7-methoxy-1,3- (23) 5-allyl-7-methoxy-1,3-benzodioxol-4-ol5-Allyl-6,7-dimethoxy (23) 66 66 5-Allyl-6,7-dimethoxy benzodioxol-4-ol (23) 66 benzodioxol-4-ol-1,3- (23)-1,3- 6677 77 5-Allyl-6,7-dimethoxy-1,3-5-Allyl-6,7-dimethoxybenzodioxol-4-ol-1,3- (23) 77 benzodioxol-4-ol-1,3- (23) 77 77 5-Allyl-6,7-dimethoxybenzodioxol-4-ol benzodioxol-4-olbenzodioxol-4-ol (30)-1,3- (30) (30) 5-Allyl-6,7-dimethoxybenzodioxol-4-ol (30) 77 5-Allyl-6,7-dimethoxybenzodioxol-4-ol -1,3-5-Allyl-6,7-dimethoxy (30) 5-Allyl-6,7-dimethoxy-1,3- 77 -1,3-5-Allyl-6,7-dimethoxybenzodioxol-4-ol (30) 77 5-Allyl-6,7-dimethoxy -1,3- 77 77 5-Allyl-6,7-dimethoxybenzodioxol-4-ol-1,3-5-Allyl-6,7-dimethoxy (30) 77 6-Allyl-2,3,4-trimethobenzodioxol-4-ol-1,3-6-Allyl-2,3,4-trimethobenzodioxol-4-ol (30)-1,3- (30) 77 6-Allyl-2,3,4-trimethoxyphenol benzodioxol-4-ol6-Allyl-2,3,4-trimetho-1,3- (30) (31) 3177 31 6-Allyl-2,3,4-trimethobenzodioxol-4-olxyphenol -1,3- xyphenol(31) (30) (31) 77 3177 benzodioxol-4-ol6-Allyl-2,3,4-trimethobenzodioxol-4-olxyphenol (30) (31) (30) 31 xyphenol benzodioxol-4-ol (31) (30) 31 6-Allyl-2,3,4-trimethobenzodioxol-4-ol xyphenol (30) (31) 6-Allyl-2,3,4-trimetho 31 6-Allyl-2,3,4-trimetho 6-Allyl-2,3,4-trimethoxyphenol 6-Allyl-2,3,4-trimetho (31) 31 31 Althoughxyphenol Pf-49487 (31)xyphenolconverted (31) methyl eugenol to compound 5 (Table 3), compound 5 was absent31 Although Pf-49487Although Pf-494876-Allyl-2,3,4-trimetho converted Pf-494876-Allyl-2,3,4-trimethoxyphenol methylconverted eugenol (31) methyl to eugenolcompound to compound 5 (Table 3), 5 compound (Table 3), compound 5 was absent31 5 was absent AlthoughAlthough Pf-49487 xyphenolconverted converted6-Allyl-2,3,4-trimetho (31) methyl methyl eugenol eugenol to compound to compound 5 (Table 5 3), (Table compound 3), compound 5 was absent31 5 was absent31 in oil preparedin oil preparedfrom6-Allyl-2,3,4-trimethoxyphenol strain from 5316,(31) xyphenolstrain which 5316, (31) expresseswhich expressesa higher a levelhigher of levelPf-49487 of , Pf-49487whereas, whereas31 inin oiloil preparedpreparedin oilAlthough frompreparedfrom strain strainPf-49487 from 5316,5316,xyphenol convertedstrain whichwhich 5316,(31) methyl expresses expresses which eugenol aexpressesa higher tohigher compound level alevel higher of5 (TableofPf-49487 Pf-49487level 3), compound,of whereas, Pf-49487whereas31 5 5-allyl-2,3-was, whereas absent 5-allyl-2,3-dimethoxyphenol5-allyl-2,3-dimethoxyphenol xyphenol (31)(Table (Table2, compound2, compound 14), compound14), compound 20, 20, dimethoxyphenol5-allyl-2,3-dimethoxyphenolAlthough5-allyl-2,3-dimethoxyphenolin oilPf-49487 prepared (Table converted2 , compoundfrom (Tablemethyl strain 14),eugenol 5316,(Table2, compound towhich compoundcompound 2,expresses 20, 5-allyl-6,7-dimethoxy-1,3-benzodioxol-4-ol 5compound (Table a14), 3),higher compound compoundlevel14), 5of was compound Pf-49487 absent20, , whereas20, Although AlthoughPf-49487 converted Pf-49487 convertedmethyl eugenol methyl to eugenol compound to compound 5 (Table 3), 5 compound(Table 3), compound 5 was absent 5 was absent in oil prepared Although from Pf-49487strain 5316, converted which methyl expresses eugenol a to higher compound level 5 (Tableof Pf-49487 3), compound, whereas 5 was absent (Table Although2, compound5-allyl-2,3-dimethoxyphenol Pf-49487 30), converted and compound methyl eugenol 31(Table were to present.compound2, These 5compound (Table results 3), compound suggest14), that 5 wascompoundPf-49487 absent does not20, in oil preparedin oilAlthough preparedfrom Pf-49487strain from 5316, convertedstrain which 5316, methyl expresses which eugenol expressesa tohigher compound alevel higher 5 (Tableof levelPf-49487 3), compoundof , Pf-49487whereas 5 ,was whereas absent 5-allyl-2,3-dimethoxyphenolin oilAlthough preparedin oil Pf-49487 preparedfrom converted strain from 5316, (Tablemethylstrain which eugenol5316, 2, expresseswhich to compoundcompound expressesa higher 5 (Table a level14),higher 3), compoundof compoundlevelPf-49487 of5 was, Pf-49487 whereas absent20,, whereas convert5-allyl-2,3-dimethoxyphenolAlthough methyl5-allyl-2,3-dimethoxyphenolin oilPf-49487Although eugenolprepared converted Pf-49487 to compoundfrom (Tablemethylconverted strain eugenol 55316, in(Table methyl2, perilla. to which eugenolcompoundcompound2, expresses to compound compound5 (Table a14), 3),higher 5compound (Table compound14),level 3), compound 5of was compound Pf-49487 absent20, 5 was , whereas absent20, in5-allyl-2,3-dimethoxyphenol oil prepared5-allyl-2,3-dimethoxyphenol from strain 5316,(Table which (Table 2,expresses compound2, a highercompound level14), of compound14),Pf-49487 , compound whereas20, 20, in oilThe prepared use in ofoil elemicin preparedfrom strain as afromsubstrate 5316, strain which resulted 5316, expresses which in lower expressesa activityhigher thanalevel higher did of the Pf-49487level use ofof, myristicinwhereasPf-49487 , (Tablewhereas3). 5-allyl-2,3-dimethoxyphenol 5-allyl-2,3-dimethoxyphenol (Table (Table2, compound2, compound 14), compound14), compound 20, 20, 5-allyl-2,3-dimethoxyphenol 5-allyl-2,3-dimethoxyphenol (Table (Table2, compound2, compound 14), compound14), Pf-49487compound 20, 20, This result suggests that myristicin and not elemicin is the main substrate for . We performed kinetic analyses of the Pf-49487 reaction using myristicin as substrate. The P450 concentration in the microsomal fraction was determined to be 1.03 µM from the reduced CO-difference spectrum and the Km, turnover number (kcat), and catalytic efficiency (kcat/Km) values were 9.1 µM, 15 min 1, and 1.65 10 6 min 1 M 1, respectively. The Km value for the oxidization of − × − − − premnaspirodiene by premnaspirodiene oxygenase (whose amino acid sequence is most similar to Pf-49487) was 14 µM, similar to that of Pf-49487 [20]. The optimum pH of Pf-49487 was approximately 7.5 (Figure7), similar to that of premnaspirodiene oxygenase [20]. Plants 2020, 9, 577 6 of 18 Plants 2020, 9, x FOR PEER REVIEW 7 of 19

A Control Myristicin

G Peak a B Pf-49487

a

C Control Methyl eugenol

H Peak b D Pf-49487

b

E Control

Elemicin

I Peak c F Pf-49487

c

FigureFigure 6. 6.Gas Gas chromatography-masschromatography- mass spectrometry spectrometry (GC- (GC-MS)MS) profiles profiles of the reaction of the reactionproducts. products.(A–F), (A–FTotal), Total ion ion chromatogramschromatograms of the of theproducts products obtained obtained using using the thecontrol control (A, (CA,E,C) ,andE) and the the microsomal microsomal fractionfraction prepared prepared from from yeast yeast harboring harboringPf-49487 Pf-49487( (BB,,DD,,F). The substrates substrates are are myristicin myristicin (A (,AB),,B methyl), methyl eugenoleugenol (C,D (C),,D and), and elemicin elemicin (E,F ().E, TheF). The controls controls were were incubated incubated without without the the cofactor cofactor NADPH. NADPH. The The peak peak with a retention time of 24 min is the internal standard, eugenol. (G–I), Mass spectra of the with a retention time of 24 min is the internal standard, eugenol. (G–I), Mass spectra of the products products formed by Pf-49487 with myristicin, methyl eugenol, and elemicine as the substrates, formed by Pf-49487 with myristicin, methyl eugenol, and elemicine as the substrates, respectively. respectively.

Plants 2020, 9, x FOR PEER REVIEW 8 of 19 PlantsPlants 2020 2020, 9, ,x 9 FOR, x FOR PEER PEER REVIEW REVIEW 8 of8 19 of 19 Plants 2020, 9, x FOR PEER REVIEW 8 of 19 Plants 2020, 9, x FOR PEER REVIEW Table 3. Relative activity of Pf-49487. 8 of 19 TableTable 3. Relative 3. Relative activity activity of Pf-49487of Pf-49487. . Plants 2020, 9, x FOR PEER REVIEW 8 of 19 FormulaTable 3. Relative activitySubstrate of Pf-49487Activity. (%) PlantsPlants 20202020, 9, ,x9 FOR, 577 PEER REVIEW 8 of7 19 of 18 FormulaTableFormula 3. Relative activitySubstrateSubstrate of Pf-49487 ActivityActivity. (%) (%) Plants 2020, 9, x FOR PEER REVIEW 8 of 19 FormulaTable 3. Relative activitySubstrate of Pf-49487Activity. (%) FormulaTable 3. Relative activitySubstrate of Pf-49487Activity. (%) FormulaTable 3. RelativeMyristicinSubstrate activity of Pf-49487Activity100 . (%) Table 3. Relative activityMyristicin of Pf-49487 . 100 Formula MyristicinSubstrate Activity100 (%) FormulaFormula MyristicinSubstrate Substrate Activity100 Activity (%) (%) Myristicin 100 Myristicin 100 Myristicin 100 MyristicinMyristicin 100 100 Methyl eugenol 91 MethylMethyl eugenol eugenol 91 91 Methyl eugenol 91

Methyl eugenol 91

Methyl eugenol 91 Methyl eugenol 91 Methyl eugenol 91 Methyl eugenol 91 Elemicin 22 ElemicinElemicin 22 22 Elemicin 22

Elemicin 22

ElemicinElemicin 22 22 Elemicin 22

Elemicin 22 Dillapiole 0 DillapioleDillapiole 0 0 Dillapiole 0

Dillapiole 0 Dillapiole 0 Dillapiole 0 Dillapiole 0 Dillapiole 0 Eugenol 0 EugenolEugenol 0 0 Eugenol 0 Eugenol 0 Eugenol 0 Eugenol 0 Eugenol 0 Eugenol 0 Limonene 0 LimoneneLimoneneLimonene 0 0 0 Limonene 0

Limonene 0

Limonene 0 Limonene 0

LimoneneGeraniolGeraniol 0 0 Geraniol 0 Geraniol 0 Geraniol 0

Geraniol 0 Geraniol 0 Geraniol 0 Geraniol 0

FigureFigure 7. pH-dependent 7. pH-dependent activity activity of Pf-49487 of Pf-49487. Data. Data are are the the means means ± SE SEof triplicate of triplicate analyses. analyses. Figure 7. pH-dependent activity of Pf-49487. Data are the means ±± SE of triplicate analyses. Figure 7. pH-dependent activity of Pf-49487. Data are the means ± SE of triplicate analyses. 3. DiscussionFigure 7. pH-dependent activity of Pf-49487. Data are the means ± SE of triplicate analyses. 3. Discussion 3. DiscussionFigure 7. pH-dependent activity of Pf-49487. Data are the means ± SE of triplicate analyses. 3. DiscussionPf-49487Figure 7.is pH-dependent a P450 enzyme activity in perilla of Pf-49487 that introduces. Data are the a means hydroxy ± SE group of triplicate onto myristicinanalyses. to form a 3. DiscussionPf-49487 is a P450 enzyme in perilla that introduces a hydroxy group onto myristicin to form a dillapiolePf-49487Figure intermediate 7. is pH-dependent a P450 enzyme (compound activity in perilla 20).of Pf-49487Pf-49487 that .introduces Datacatalyzes are the a means thehydroxy hydroxylation ± SE group of triplicate onto of methylanalyses.myristicin eugenol to form and a 3.dillapiole DiscussionPf-49487Figure intermediate is 7.a P450pH-dependent enzyme(compound activityin perilla 20). of Pf-49487 Pf-49487that introduces .catalyzes Data are a the thehydroxy means hydroxylation ± group SE of triplicate onto of methylmyristicin analyses. eugenol to form and a 3. DiscussionelemicindillapiolePf-49487 at intermediate ais positiona P450 enzyme similar (compound toin thatperilla of20). compoundthat Pf-49487 introduces catalyzes 20 but a ishydroxy unreactivethe hydroxylation group towards onto ofmyristicin limonene methyl and eugenolto form geraniol, anda dillapioleelemicin atintermediate a position (compoundsimilar to that 20). ofPf-49487 compound catalyzes 20 but the ishydroxylation unreactive towards of methyl limonene eugenol and 3.dillapiole DiscussionelemicinPf-49487 intermediate at is a a positionP450 enzyme(compound similar in perillato 20). that Pf-49487 that of compoundintroduces catalyzes a20 thehydroxy but hydroxylation is unreactivegroup onto of towards methylmyristicin eugenollimonene to form and aand elemicin3. DiscussionwhichPf-49487 areat a MT-type isposition a P450 oil enzymesimilar components, into perillathat asof that wellcompound introduces as dillapiole 20 abut hydroxy and is eugenol,unreactive groupwhich onto towards myristicin are phenylpropanoidlimonene to form and a dillapioleelemicin atintermediate a position (compoundsimilar to that 20). ofPf-49487 compound catalyzes 20 but the ishydroxylation unreactive towards of methyl limonene eugenol and dillapiolecompounds. Pf-49487 intermediate is a These P450 resultsenzyme(compound indicate in perilla 20). that Pf-49487 thatPf-49487 introduces catalyzesexhibits a thehydroxy rather hydroxylation group low substrate onto of methylmyristicin specificity eugenol to form and and high a elemicin Pf-49487 at a isposition a P450 enzymesimilar toin perillathat of that compound introduces 20 abut hydroxy is unreactive group onto towards myristicin limonene to form and a dillapiole elemicinregiospecificity. atintermediate a position Premnaspirodiene (compoundsimilar to that 20). oxygenase, ofPf-49487 compound catalyzes whose 20 sequencebutthe ishydroxylation unreactive is most similartowards of methyl to limonene that eugenol of Pf-49487 and , elemicindillapiole at intermediate a position (compoundsimilar to that 20). ofPf-49487 compound catalyzes 20 but the ishydroxylation unreactive towards of methyl limonene eugenol and elemicin at a position similar to that of compound 20 but is unreactive towards limonene and

Plants 2020, 9, 577 8 of 18 has similar properties [20]. Other members of the CYP71D family also have high regiospecificity, namely the limonene hydroxylases CYP71D13 and CYP71D18, which hydroxylate the C3 and C6 positions, respectively [21]. The biosynthetic pathways of PP-type oil components have been inferred from the results of crossing experiments conducted with pure strains of perilla containing elemicin, myristicin, dillapiole, and nothoapiole as the main oil components, and the genetic control of each step in the formation of elemicin, dillapiole, and nothoapiole was in accordance with Mendelism [16] (Figure1). Phenylpropanoids are believed to be synthesized via the shikimate pathway and the genes for methyl eugenol formation have been cloned [22]; consequently, methyl eugenol could be a precursor of PP-type oil components. However, neither methyl eugenol nor compound 5 have been detected in perilla oil whereas Pf-49487 produces compound 5 from methyl eugenol in vitro. On the other hand, compound 14 (a putative precursor of elemicin and myristicin) and compound 30 (a putative precursor of nothoapiole) were present in oil from strain 5316 (PP-mdn). However, compound 14 was not found in oil from strain 10 (PP-em) and strain 12 (PP-m), even though compound 14 is a likely intermediate of myristicin and thus was expected to be present in these oils. These findings indicate that the catalytic patterns and reactivities of enzymes may differ among oil types. Our identification of monohydrate intermediates of phenylpropanoid compounds in perilla oil suggests that O-methyltransferases or P450s are involved in perilla biosynthetic pathways and catalyze the generation of methoxy groups or methylenedioxy bridges from these monohydrate compounds. Our future studies will further explore these enzymes.

4. Materials and Methods

4.1. Plant Materials All perilla plants used in this study were grown at the Experimental Station for Medicinal Plant Research, Graduate School of Pharmaceutical Sciences, Kyoto University. They have been bred and kept as pure lines through repeated self-pollination [18]. Strain numbers and oil types (Figure1) of perilla used in this study were as follows: strain 10, PP-em; strain 12, PP-m; strain 16, PP-md; strain 25, PP-emd; strain 5316, PP-mdn; strain 5717, C.

4.2. Construction of the Expressed Sequence Tag (EST) Library and Cloning of Pf-49487 The EST library was constructed by the Kazusa DNA Research Institute as described previously [7]. P450-like sequences were selected from the EST library by BLAST analysis and the expression levels of these sequences were compared between different oil types. High expression level contigs in strains containing high levels of dillapiole (strains 16, 25, and 5316) were selected and their expression levels were compared using RPKM values. We focused on contig 49487, which is most specifically expressed in dillapiole-containing perilla (Table1). PCR-amplification was performed for the target sequence, using cDNA as a template. cDNA was obtained by reverse transcription of RNA isolated from fresh young perilla leaves (strain 5316) using a RNeasy Plant Mini Kit (Qiagen), and reverse transcription was performed using RevTra Ace (Toyobo) with primer add2 (50-CCACGCGTCGACTACTTTTTTTTTTTTTTT-30). Primers for PCR-amplification were designed based on sequence information from the EST database. The forward primer 49487-f1 (50-TCCGTTCCGTTCCTTCAGAGATCTCGCG-30) and reverse primer 49487-r1 (50-GGATGCCTTATCAGTTCAGTCATTGCC-30) were used for amplifying contig 49487. 1 The reaction mixture contained 0.2 µM primer, 0.2 mM dNTPs, and 0.025 U µL− Blend Taq (Toyobo). The temperature program started at 94 ◦C for 30 s, followed by 30 cycles of 51 ◦C for 30 s, 72 ◦C for 60 s, and a final elongation at 72 ◦C for 60 s. The size of contig 49487 corresponded to the sequence information from the EST database and RACE methods were used to obtain its full-length sequence. 30-RACE was performed with the primers add2 and 49487-f2 (50-AGAAGGTCGGCACAATGGTCAGC TCCATC-30), and then nested PCR was performed with the primers amm (50-GGCCACGCGTCGACTAC-30) Plants 2020, 9, 577 9 of 18

and 49487-f3 (50-TGTAGGTCTGCGTTCGGCACGGTGTGCAAG-30) in the same reaction mixture as described above, with a temperature program starting at 94 ◦C for 30 s, followed by 30 cycles of 55 ◦C (52 ◦C for nested PCR) for 30 s, 72 ◦C for 60 s, and a final elongation at 72 ◦C for 60 s. The reaction products were electrophoresed in agarose gel, purified using NucleoSpin Gel and PCR clean-up (Macherey-Nagel), and ligated to the vector pTA2 (Toyobo). Sequences were confirmed using FASMAC. For 50-RACE, reverse transcription was performed as described above with primer 49487-r2 (50-GTAATTGTACCAGCAGACCTCTAGG-30) or primer 49487-r3 (50-GGATCATTGTCTTTGAGGACT TCCTTC-30) and the reaction products were purified using NucleoSpin Gel and PCR clean-up. After the 1 addition of poly C using 0.6 U µL− TdT (Invitrogen) and 0.2 mM dCTP (Toyobo), PCR was performed with the primers 5ann (50-GGCCACGCGTCGACTAGTACGGG(I)(I)GGG(I)(I)GGG(I)(I)G-30) and 49487-r3 or 49487-r4 (50-CGATGACACGAGGAACGAGTCGAC-30), and then nested PCR was performed with the primers amm and 49487-r3 in the same reaction mixture as described above, with a temperature program starting at 94 ◦C for 100 s, followed by 25 cycles (30 cycles for nested PCR) of 94 ◦C for 30 s, 50 ◦C (50.5 ◦C for nested PCR) for 30 s, 72 ◦C for 60 s, and a final elongation at 72 ◦C for 60 s.

4.3. Heterologous Expression of Pf-49487 in Saccharomyces cerevisiae Pf-49487 was ligated into yeast expression vector pGYR-SpeI containing the S. cerevisiae NADPH-P450 reductase gene and a SpeI-cloning site [17]. The full-length sequence of Pf-49487 was amplified by PCR with the forward primer 49487-f4 (50-ACTAGTATGGAGTCCGATCTCGCAACTG-30) and the reverse primer 49487-r5 (50-ACTAGTTCACGGTGATGTCGGTTCAAATGG-30) in a reaction 1 mixture containing 0.3 µM primer, 0.2 mM dNTPs, and 0.02 U µL− KOD-Plus (Toyobo) using a temperature program starting at 94 ◦C for 100 s, followed by 25 cycles of 94 ◦C for 15 s, 52.7 ◦C for 30 s, 68 ◦C for 90 s, and final elongation at 68 ◦C for 90 s. The amplified sequence was ligated into pTA2 and the nucleotide sequence was confirmed, then Pf-49487 was ligated into pGYR-SpeI and the vector was introduced into S. cerevisiae strain AH22 using the LiCl method. The recombinant yeast cells were cultivated, and the microsomal fraction was prepared as previously described [23]. The microsomal fraction was suspended in 100 mM HEPES/NaOH (pH 7.5) and stored at 80 C − ◦ until needed. The reduced CO-difference spectrum was measured with a UV1800 spectrophotometer (Shimadzu) and enzymatic activity was confirmed by the peak at 450 nm [24].

4.4. Enzymatic Assays and Gas Chromatography-Mass Spectrometry (GC-MS) Analyses Enzymatic reactions were performed in 1-mL volumes in screw-capped glass tubes. Each reaction mixture was composed of 50 mM HEPES/NaOH (pH 7.5), 0.5 mM NADPH, 0.2 mM substrate, and 1 1.98 mg mL− enzyme preparation. After incubation at 30 ◦C for 15 min, 12 nmol of eugenol was added as an internal standard, and the reaction mixture was extracted three times with 2 mL of pentane. The pentane fractions were combined, dehydrated with MgSO4, concentrated under nitrogen, and analyzed by gas chromatography-mass spectrometry (GC-MS) instrument (6850GC/5975MSD, Agilent Technologies or GCMS-QP2020 NX, Shimadzu). The compounds were separated on a DB-WAX column (60 m 0.25 mm 0.25 µm, Agilent Technologies, Santa Clara, CA, USA) under the following × × 1 conditions: injector, 180 ◦C; oven program starting at 100 ◦C, increasing at 5 ◦C min− to 220 ◦C, and holding at this temperature for 60 min. Helium was used as the carrier gas and column flow was 1 1.0 mL min− . The compounds were identified by comparing their retention times and mass spectra with authentic standards, synthesized samples, or a MS data library (NIST11 or NIST17; National Institute of Standards and Technology). 1 For kinetic analyses, 0.78 mg mL− enzyme preparations were incubated with myristicin concentrations ranging from 0.2 to 10 µM. The reaction mixtures were treated as described above and analyzed by GC-FID (G5000, Hitachi, Tokyo, Japan). Compounds were separated on an InertCap WAX column (60 m 0.25 mm 0.25 µm, GL Sciences) or a DB-WAX column (60 m 0.25 mm 0.25 µm, × × × × Agilent Technologies) under the following conditions: injector, 180 ◦C; FID, 220 ◦C; oven program Plants 2020, 9, 577 10 of 18

1 starting at 100 ◦C, increasing at 5 ◦C min− to 220 ◦C, and holding at this temperature for 40 min. Assays were repeated independently three times. The P450 concentration in the enzyme preparation was determined from the reduced CO-difference spectrum using a differential absorption coefficient of 1 1 91 mM− cm− [24]. 1 The optimum pH (ranging from 6.0 to 9.0) was analyzed using 0.2 mM myristicin and 1.65 mg mL− enzyme preparation. Assays were performed in the same manner as the kinetic analyses.

4.5. Quantitative RT-PCR The differences in the expression levels of Pf-49487 between different perilla oil types were determined using quantitative RT-PCR. Total RNA was isolated from fresh young perilla leaves (strains 12, 25, and 5316) using the method described above. First-strand cDNAs were synthesized from 1 µg total RNA using ReverTra Ace and oligo(dT) primer (Takara), and were then purified using NucleoSpin Gel and PCR clean-up. Quantitative RT-PCR was performed with StepOnePlus (Applied Biosystems) using THUNDERBIRD SYBR qPCR Mix (Toyobo) following the manufacturers’ protocols, with a temperature program starting at 95 ◦C for 60 s, followed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for 60 s. The forward primer 49487-f (50-TGGTGCCTCTCATAATGCTG-30) and the reverse primer 49487-r (50-TCTGAAGGAACGGAACGGAG-30) were used to amplify Pf-49487, and the forward primer Histone-f (50-TCACGAACAAGCCTTTGGAA-30) and the reverse primer Histone-r (50-AAGCCTCACCGTTACCGTC-30) were used to provide histone transcripts as the internal control ∆∆CT for PCR. Quantitation was performed using the 2− method. Total RNA was extracted in duplicate and reactions were performed independently three times.

4.6. GC-MS Analysis of Perilla Oil Perilla essential oils were obtained by extracting approximately 300 g of fresh perilla leaves (strains 10, 12, and 5316) with diethyl ether overnight at 4 ◦C. The oils were concentrated, dehydrated, and analyzed by GC-MS as described above. Because these analyses were performed to confirm the presence of monohydrate intermediates, the oils were highly concentrated; consequently, we focused on the retention times of the synthesized compounds (Table2). The chemical profiles of each perilla strain were previously reported [4,16].

4.7. Chemicals Chemical reagents were purchased from Nacalai Tesque or Fujifilm Wako Pure Chemical. Compounds 5, 8, 14, 20, 23, and 30 were synthesized as described below (Figure8). NMR, MS, and IR data are shown in Supplementary Data S1. Compound 31 was synthesized as previously reported [25]. Plants 2020, 9, 577 11 of 18 Plants 2020, 9, x FOR PEER REVIEW 12 of 19

FigureFigure 8. Synthetic 8. Synthetic scheme scheme of allylphenols. of allylphenols. (A) Synthesis (A) Synthesis of 6-allyl-2,3-dimethoxyphenol of 6-allyl-2,3-dimethoxyphenol (5). (B) Alternative(5). (B) synthesisAlternative of 6-allyl-2,3-dimethoxyphenol synthesis of 6-ally (5). (l-2,3-dimethoxyphenolC) Synthesis of 2-allyl-4,5-dimethoxyphenol (5). (C) Synthesis (8). (D) Synthesisof of 5-allyl-2,3-dimethoxyphenol2-allyl-4,5-dimethoxyphenol (8). (14). (D) Synthesis (E) Synthesis of 5-allyl-2,3-dimethoxyphenol of 6-allyl-4-methoxy-1,3-benzodioxol-5-ol (14). (E) Synthesis of (20). (F) Synthesis6-allyl-4-methoxy-1,3-benzodioxol-5-ol of 5-allyl-7-methoxy-1,3-benzodioxol-4-ol (20). (F) Synthesis (23). of ( G5-allyl-7-methoxy-1,3-benzodioxol-4-ol) Synthesis of 5-allyl-6,7-dimethoxy-1,3- benzodioxol-4-ol(23). (G) Synthesis (30). of 5-allyl-6,7-dimethoxy-1,3-benzodioxol-4-ol (30).

Plants 2020, 9, 577 12 of 18

All melting points were uncorrected. Silica gel was used for column chromatography. Commercially available solvents and reagents were purchased and used without purification.

4.7.1. Synthesis of 6-Allyl-2,3-dimethoxyphenol (5) 1. 2-Benzenesulfonyl-1,3-dibromopropane [219500-61-5] (1): The title compound was prepared according to the reported procedure as colorless needles [26,27] of mp 93–94 ◦C (EtOAc/hexane). 2. 1-(2-Benzenesulfonylallyloxy)-2,3-dimethoxybenzene (2): 2,3-Dimethoxyphenol (262 mg, 1.70 mmol) was dissolved in dry acetone (17 mL), and to the solution was added K2CO3 (705 mg, 5.10 mmol). The resulting suspension was stirred at room temperature for 10 min, and 2-benzenesulfonyl-1,3-dibromopropane (1) (640 mg, 1.87 mmol) and KI (7 mg, 0.04 mmol) were added to the suspension. After 3 h, the mixture was filtered through Celite, and the residue was successively washed with EtOAc. The combined filtrate and washings were concentrated in vacuo and purified by column chromatography (hexane/EtOAc 4:1) to give 2,3-dimethoxyphenol (45 mg, 17%) and the title compound (453 mg, 80%) as pale-yellow oils. 3. 6-(2-Benzenesulfonylallyl)-2,3-dimethoxyphenol (3): 1-(2-Benzenesulfonylallyloxy)-2,3- dimethoxybenzene (2) (436 mg, 1.30 mmol) was dissolved in toluene (1 mL) and heated at 165 ◦C for 12 h under microwave irradiation. The mixture was concentrated in vacuo and purified by column chromatography (hexane/EtOAc 7:3) to give the title compound (398 mg, 92%) as a pale-yellow solid: mp 108–109 ◦C (EtOAc/hexane). 4. 3-Benzenesulfonyl-7,8-dimethoxychromane (4): To a solution of 6-(2-benzenesulfonylallyl)- 2,3-dimethoxyphenol (3) (144 mg, 0.430 mmol) in dry acetone (4 mL), was added K2CO3 (89 mg, 0.64 mmol), and the mixture was heated under reflux for 5 h. The mixture was diluted with EtOAc and washed with H2O. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo to give the title compound as a white solid (140 mg). The crude product was used in the next step without further purification. The title compound was characterized after recrystallization from EtOAc/hexane, which gave colorless needles of mp 132–134 ◦C. 5. 6-Allyl-2,3-dimethoxyphenol [450357-58-1] (5): To a solution of the crude 3-benzenesulfonyl-7,8- dimethoxychromane (4) (140 mg) in EtOAc/MeOH (2:1, 4.5 mL), was added 5% sodium amalgam (0.93 g, 2.0 mmol) at room temperature, and the mixture was stirred for 4 h. The reaction was quenched by the addition of solid citric acid (0.39 g), and the mixture was diluted with EtOAc, washed with saturated aqueous NaHCO3, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 9:1) to give the title compound (65 mg, 78% over 2 steps) as a pale-yellow oil.

4.7.2. Alternative Synthesis of 6-Allyl-2,3-dimethoxyphenol (5) 1. 1-(Allyloxy)-2,3-dimethoxybenzene [380621-78-3] (6): The title compound was prepared from 2,3-dimethoxyphenol (982 mg, 6.37 mmol) according to the reported procedure [28]. After purification by column chromatography (hexane/EtOAc 9:1), the title compound (1.11 g, 90%) was obtained as a colorless oil. 2. 6-Allyl-2,3-dimethoxyphenol [450357-58-1] (5) and 4-Allyl-2,3-dimethoxyphenol [29445-64-5] (7): A solution of 1-allyloxy-2,3-dimethoxtbenzene (6) (528 mg, 2.72 mmol) in decaline (0.5 mL) was heated at 200 ◦C under microwave irradiation for 10 h. After cooled to ambient temperature, the solution was directly purified by column chromatography (hexane/EtOAc 9:1) to give 6-allyl-2,3-dimethoxyphenol (103 mg, 20%) as a pale-yellow oil and a 63:37 mixture of 6-allyl-(5) and 4-allyl-2,3-dimethoxyphenol (7) (367 mg, 44% and 26%, respectively) as a pale-yellow oil. 4-Allyl-2,3-dimethoxyphenol (7) was characterized as a mixture with the other isomer. Plants 2020, 9, 577 13 of 18

4.7.3. Synthesis of 2-Allyl-4,5-dimethoxyphenol (8) 2-Allyl-4,5-dimethoxyphenol [59893-87-7] (8) and 2-Allyl-3,4-dimethoxyphenol [66967-26-8] (9): 4-allyloxy-1,2-dimethoxybenzene [29] (515 mg, 2.65 mmol) was dissolved in decaline (0.5 mL) and heated at 200 ◦C under microwave irradiation for 12 h. After cooled to ambient temperature, the solution was directly purified by column chromatography (hexane/EtOAc 3:1) to give 2-allyl-4,5-dimethoxyphenol (8) (68 mg, 13%) as a pale-yellow solid along with a 91:9 mixture of the title compounds (411 mg, 73% and 7%, respectively) as a pale-yellow oil. 2-Allyl-3,4-dimethoxyphenol (9) was partially separated as a pale-yellow oil from the mixture by another column chromatography (hexane/EtOAc 3:1) for characterization. 2-Allyl-4,5-dimethoxyphenol: mp 35–36 ◦C (Et2O/hexane) (lit. 42–42.5 ◦C) [30].

4.7.4. Synthesis of 5-Allyl-2,3-dimethoxyphenol (14) 1. 3-tert-Butyldimethylsiloxy-4,5-dimethoxybenzaldehyde [122271-47-0] (10): To a solution of 3-hydroxy-4,5-dimethoxybenzaldehyde (500 mg, 2.74 mmol) in dry N,N-dimethylformamide (DMF) (3 mL), were added tert-butylchlorodimethylsilane (496 mg, 3.29 mmol) and imidazole (466 mg, 6.84 mmol). The mixture was stirred at room temperature for 16 h, diluted with EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo. The resulting pale-yellow oil, containing mainly the title compound, was used for the next step without further purification. The 1H NMR was consistent with that reported [31]. 2. 3-tert-Butyldimethylsiloxy-4,5-dimethoxybenzenemethanol [111394-55-9] (11): To a solution of the crude 3-tert-butyldimethylsiloxy-4,5-dimethoxybenzaldehyde (10) in EtOH (24 mL) cooled in an ice–water bath, was portion-wise added NaBH4 (104 mg, 2.75 mmol). After 15 min, water was added to the mixture, and most of EtOH was removed from the mixture by evaporation. The mixture was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography (hexane/EtOAc 3:1) to give the title compound (563 mg, 69% over 2 steps) as a white solid. Recrystallization from hexane gave colorless needles of mp 57–58 ◦C. The 13C NMR was identical to that reported, while all the 1H NMR chemical shifts differed by 0.18 ppm from the reported values [32]. 3. 3-tert-Butyldimethylsiloxy-4,5-dimethoxybenzyl Bromide [111394-56-0] (12): To a solution of 3-tert-butyldimethylsiloxy-4,5-dimethoxybenzenemethanol (11) (315 mg, 1.06 mmol) in dry CH2Cl2 (10 mL) cooled in an ice–water bath, were added CBr4 (420 mg, 1.27 mmol) and Ph3P (333 mg, 1.27 mmol). The cooling bath was removed, and the mixture was stirred at room temperature for 20 min. Volatile materials were removed from the mixture by evaporation, and the residue was purified by column chromatography (hexane to hexane/EtOAc 97:3) to give the title compound (305 mg, 80%) as a pale-yellow oil. 4. 5-Allyl-1-tert-butyldimethylsiloxy-2,3-dimethoxybenzene (13): To a mixture of 3-tert- butyldimethylsiloxy-4,5-dimethoxybenzyl bromide (12) (396 mg, 1.10 mmol), CuI (21 mg, 0.11 mmol), and 2,2’-bipyridine (17 mg, 0.11 mmol) in Et2O (1 mL) cooled in an ice–water bath, was dropwise added a 1.0 M tetrahydrofuran (THF) solution of vinylmagnesium bromide (1.6 mL, 1.6 mmol). The cooling bath was removed, and the mixture was stirred at room temperature for 1 h. The reaction was quenched by the addition of saturated aqueous NH4Cl and then 28% aqueous NH3. After stirred for 30 min, the whole was extracted three times with Et2O. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 49:1) to give the title compound (266 mg, 78%) as a pale-yellow oil. 5. 5-Allyl-2,3-dimethoxyphenol [76773-99-4] (14): To a stirred solution of 5-allyl-1-tert- butyldimethylsiloxy-2,3-dimethoxybenzene (13) (74 mg, 0.24 mmol) in THF (1 mL), was added a 1.0 M THF solution of tetrabutylammonium fluoride (TBAF) (0.24 mL, 0.24 mmol). After 30 min, 0.5 M aqueous citric acid (1 mL) was added to the mixture, and the whole was extracted three Plants 2020, 9, 577 14 of 18

times with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 9:1) to give the title compound (43 mg, 91%) as a pale-yellow solid of mp 38–40 ◦C.

4.7.5. Synthesis of 6-Allyl-4-methoxy-1,3-benzodioxol-5-ol (20) 1. 4-Methoxy-1,3-benzodioxole [1817-95-4] (15): The title compound was prepared from 3-methoxycatechol according to the reported procedure [33] in 85% yield as colorless blocks of mp 41–42 ◦C. 2. 4-Methoxy-1,3-benzodioxole-5-carbaldehyde [5779-99-7] (16) and 7-Methoxy-1,3-benzodioxole-4- carbaldehyde [23731-55-7] (17): Dry DMF (0.64 mL, 8.3 mmol) and freshly distilled POCl3 (0.77 mL, 8.3 mmol) were mixed at 100 ◦C for 2 h. To the mixture, 4-methoxy-1,3-benzodioxole (15) (500 mg, 3.29 mmol) was added, and the mixture was heated at 100 ◦C. After 2 h, the reaction was quenched by the addition of ice, and the whole was extracted three times with Et2O. The combined organic layers were washed with water, saturated aqueous NaHCO3, and brine, dried over Na2SO4, and concentrated in vacuo to give a crude mixture of the title compounds (3:2) as a pale brown solid. The two isomers were separated by column chromatography (hexane/Et2O 4:1, Rf = 0.24 and 0.12, respectively). 4-Methoxy-1,3-benzodioxole-5-carbaldehyde (16): 44% yield. Colorless needles of mp 107–108 ◦C (EtOH). 7-Methoxy-1,3-benzodioxole-4-carbaldehyde (17): 29% yield. Colorless needles of mp 85–86 ◦C (EtOH). 3. 4-Methoxy-1,3-benzodioxol-5-ol [23504-78-1] (18): To a solution of 4-methoxy-1,3-benzodioxole- 5-carbaldehyde (16) (100 mg, 0.555 mmol) in MeOH (1 mL), were added conc. H2SO4 (0.01 mL, 0.2 mmol) and 30% H2O2 (0.085 mL, 0.83 mmol). The mixture was stirred at room temperature for 2.5 h, and saturated aqueous NaHCO3 was added to the mixture. The whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual solid was purified by column chromatography (hexane/EtOAc 9:1) to give the title compound (72 mg, 78%) as a white solid: Rf = 0.29 (hexane/EtOAc 4:1). Colorless plates of mp 58–59 ◦C (Et2O/hexane). 4. 5-Allyloxy-4-methoxy-1,3-benzodioxole [23731-59-1] (19): A mixture of 4-methoxy-1,3- benzodioxol-5-ol (18) (67 mg, 0.40 mmol), K2CO3 (111 mg, 0.800 mmol), and allyl bromide (0.05 mL, 0.6 mmol) in dry acetone (4 mL) was heated under reflux for 4.5 h, and water was added to the mixture. The whole was extracted three times with EtOAc, and the combined organic layers were washed with 15% aqueous NaOH twice and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 95:5) to give the title compound (71.6 mg, 86%) as a pale-yellow oil: Rf = 0.25 (hexane/EtOAc 19:1). 5. 6-Allyl-4-methoxy-1,3-benzodioxol-5-ol [23731-60-4] (20): A solution of 5-allyloxy-4-methoxy- 1,3-benzodioxole (19) (58 mg, 0.28 mmol) in a 1:2 mixture of N-methyl-2-pyrrolidone (NMP) and decaline (1.5 mL) was heated at 200 ◦C under microwave irradiation for 8 h. After cooled to ambient temperature, the mixture was diluted with EtOAc, washed three times with water and with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 9:1) to give the title compound (46 mg, 78%) as a pale-yellow oil: Rf = 0.44 (hexane/EtOAc 4:1).

4.7.6. Synthesis of 5-Allyl-7-methoxy-1,3-benzodioxol-4-ol (23) 1. 7-Methoxy-1,3-benzodioxol-4-ol [23812-54-6] (21): To a solution of 7-methoxy-1,3-benzodioxole- 4-carbaldehyde (17) (100 mg, 0.555 mmol) in MeOH (1 mL), conc. H2SO4 (0.01 mL, 0.2 mmol) and 30% H2O2 (0.085 mL, 0.83 mmol) were added. The mixture was stirred at room temperature for 6 h, and saturated aqueous NaHCO3 was added to the mixture. The whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and Plants 2020, 9, 577 15 of 18

concentrated in vacuo. The residual solid was purified by column chromatography (hexane/EtOAc 3:1) to give the title compound (85 mg, 92%) as a white solid: Rf = 0.13 (hexane/EtOAc 4:1). Colorless needles of mp 104–105 ◦C (CHCl3/hexane). 2. 4-Allyloxy-7-methoxy-1,3-benzodioxole [23731-70-6] (22): A mixture of 7-methoxy-1,3- benzodioxol-4-ol (21) (82 mg, 0.49 mmol), K2CO3 (135 mg, 0.977 mmol), and allyl bromide (63 µL, 0.73 mmol) in dry acetone (2.5 mL) was heated under reflux for 3 h, and water was added to the mixture. The whole was extracted three times with EtOAc, and the combined organic layers were washed with 15% aqueous NaOH twice and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 95:5) to give the title compound (83.7 mg, 82%) as a pale-yellow oil: Rf = 0.20 (hexane/EtOAc 19:1). 3. 5-Allyl-7-methoxy-1,3-benzodioxol-4-ol [76773-99-4] (23): A solution of 4-allyloxy-7-methoxy- 1,3-benzodioxole (22) (82 mg, 0.39 mmol) in a 1:4 mixture of NMP and decaline (1 mL) was heated at 200 ◦C under microwave irradiation for 8 h. After cooled to ambient temperature, the mixture was diluted with EtOAc, washed three times with water and with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane/EtOAc 4:1) to give the title compound (68 mg, 84%) as a pale-yellow solid: Rf = 0.24 (hexane/EtOAc 4:1). Colorless needles of mp 84–85 ◦C (EtOAc/hexane).

4.7.7. Synthesis of 5-Allyl-6,7-dimethoxy-1,3-benzodioxol-4-ol (30) 1. 6,7-Dimethoxy-1,3-benzodioxol-4-ol [22934-71-0] (24): A mixture of 6,7-dimethoxy-1,3-benzodioxole- 4-carbaldehyde (368 mg, 1.75 mmol), 30% H2O2 (0.27 mL, 2.6 mmol), and conc. H2SO4 (0.12 mL, 2.3 mmol) in MeOH (3.5 mL) was stirred at room temperature for 3 h. After addition of saturated aqueous NaHCO3, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual pale-yellow solid (1.38 g, 78%) was used as a crude title compound without further purification to the next step.: Rf = 0.22 (hexane/EtOAc 2:1). Colorless needles of mp 111–112 ◦C (EtOAc/hexane). 2. 4-Hydroxy-6,7-dimethoxy-1,3-benzodioxol-5-carbadehyde (25): A mixture of 6,7-dimethoxy-1,3- benzodioxol-4-ol (24) (589 mg, 2.97 mmol) and hexamethylenetetramine (834 mg, 5.94 mmol) in refluxing trifluoroacetic acid (TFA) (3 mL) was stirred for 24 h [34]. H2O was added, and the solution was stirred for further 30 min at 60 ◦C. After being cooled to room temperature, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual solids were purified by column chromatography (hexane/EtOAc 7:1) to give the title compound (546 mg, 81%) as a pale-yellow solid: Rf = 0.25 (hexane/EtOAc 7:1). Colorless powder of mp 85–87 ◦C (EtOAc/hexane). 3. 4-tert-Butyldimethylsilyloxy-6,7-dimethoxy-1,3-benzodioxole-5-carbadehyde (26): To a mixture of 4-hydroxy-6,7-dimethoxy-1,3-benzodioxole-5-carbaldehyde (25) (1.20 g, 5.31 mmol), 4-dimethylaminopyridine (DMAP) (130 mg, 1.06 mmol), and Et3N (1.5 mL, 11 mmol) in CH2Cl2 (27 mL), was added tert-butylchlorodimethylsilane (1.20 g, 7.96 mmol). The mixture was stirred at room temperature for 3 h. After addition of saturated aqueous NaHCO3, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual pale-yellow oil (2.14 g) was used as a crude title compound without further purification to the next step. 4. 4-tert-Butyldimethylsilyloxy-6,7-dimethoxy-1,3-benzodioxole-5-methanol (27): To a solution of the crude 4-tert-butyldimethylsiloxy-6,7-dimethoxy-1,3-benzodioxole-5-carbaldehyde (26) (2.14 g) in EtOH (30 mL) was added NaBH4 (714 mg, 18.8 mmol). The mixture was stirred at room temperature for 30 min. After addition of water, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual solids were purified by column chromatography (hexane/EtOAc 9:1 to 5:1) to give the title compound (1.73 g, 95% in two steps) as a colorless oil: Rf = 0.23 (hexane/EtOAc 7:1). Plants 2020, 9, 577 16 of 18

5. 5-Bromomethyl-4-tert-butyldimethylsilyloxy-6,7-dimethoxy-1,3-benzodioxole (28): To a mixture of 4-tert-butyldimethylsiloxy-6,7-dimethoxy-1,3-benzodioxole-5-methanol (27) (450 mg, 1.31 mmol) in CH2Cl2 (13 mL) was added PBr3 (0.14 mL, 1.4 mmol). The mixture was stirred at room temperature for 30 min. After addition of water, the whole was extracted three times with CHCl3, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual pale-yellow oil (552 mg) was used as a crude title compound without further purification to the next step. 6. 5-Allyl-4-tert-butyldimethylsilyloxy-6,7-dimethoxy-1,3-benzodioxole (29): To a mixture of the crude 5-bromomethyl-4-tert-butyldimethylsiloxy-6,7-dimethoxy-1,3-benzodioxole (28) (552 mg, 1.56 mmol), CuI (50 mg, 0.26 mmol), and 2,2’-bipyridine (41 mg, 0.26 mmol) in Et2O (4 mL) was added a 1.0 M THF solution of vinylmagnesium bromide (2.0 mL, 2.0 mmol). The mixture was stirred at room temperature for 30 min. After addition of water, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual solids were purified by column chromatography (hexane/EtOAc 50:1) to give the title compound (313 mg, 68% over 2 steps) as a pale-yellow oil: Rf = 0.52 (hexane/EtOAc 7:1). 7. 5-Allyl-6,7-dimethoxy-1,3-benzodioxol-4-ol (30): To a solution of 5-allyl-4-tert-butyldimethylsiloxy- 6,7-dimethoxy-1,3-benzodioxole (29) (690 mg, 1.96 mmol) in THF (20 mL) was added a 1.0 M THF solution of TBAF (1.96 mL, 2.0 mmol). The mixture was stirred at room temperature for 30 min. After addition of water, the whole was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residual solids were purified by column chromatography (hexane/EtOAc 5:1 to 4:1) to give the title compound (462 mg, 99%) as a pale-yellow solid: Rf = 0.28 (hexane/EtOAc 4:1). Colorless needles of mp 65–67 ◦C (EtOAc/hexane).

5. Conclusions An enzyme catalyzing a reaction in a proposed synthetic pathway of PP-type perilla oil components, namely, the conversion of myristicin into a monohydrate intermediate of dillapiole (compound 20) by regiospecific hydroxylation, was cloned. The potentially harmful to humans (E)-Asarone is structurally similar to PP-type oil components and has been identified in perilla plants grown in China [12,13]. The methoxy group of (E)-asarone may result from the hydroxylation of an intermediate. The position of the double bond in the side chain of (E)-asarone differs from that of PP-type oil components and thus Pf-49487 is unlikely to be involved in the biosynthesis of (E)-asarone. However, our findings regarding Pf-49487 may help elucidate the (E)-asarone biosynthetic pathway and aid in the quality assessment and safety evaluation of perilla products. Little is known about the biosynthetic pathways of phenylpropanoid volatile components. Only a few enzymes involved in the synthesis of compounds such as anethole and eugenol have been reported, and enzymes involved in the synthesis of compounds such as apiole, myristicin, and asarone have not been characterized. These components are important plant flavor compounds and are also used as medicines and food supplements due to their pharmacological actions. Some of these compounds are genotoxic or carcinogenic [8]. Further elucidation of the biosynthetic mechanisms of phenylpropanoid volatile components may contribute to the genetic manipulation and enzymatic production of useful compounds.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/5/577/s1, Supplementary Data S1: Analytical data for the synthesized compounds. Author Contributions: Conceptualization and project administration, M.I.; investigation and writing—original draft preparation, M.B.; methodology and writing—original draft preparation of the organic syntheses, K.-i.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Plants 2020, 9, 577 17 of 18

Acknowledgments: We thank David R. Nelson of the Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, for naming the sequence of Pf-49487 according to the standardized P450 nomenclature system. We thank Toshiyuki Sakaki of the Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, for providing the expression vector pGYR-SpeI and S. cerevisiae strain AH 22. We thank Yousuke Yamaoka of the Department of Synthetic Medical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, for his guidance and assistance with organic synthesis. Conflicts of Interest: The authors declare no conflict of interest.

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