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Plant Biotechnol. 37(3) Plant Biotechnology 37, 301–310 (2020) DOI: 10.5511/plantbiotechnology.20.0421a Original Paper Molecular cloning and biochemical characterization of isoflav-3-ene synthase, a key enzyme of the biosyntheses of (+)-pisatin and coumestrol Kai Uchida1, Toshio Aoki1,a, Hideyuki Suzuki2, Tomoyoshi Akashi1,* 1 Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan; 2 Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan * E-mail: [email protected] Tel & Fax: +81-466-84-3353 Received March 27, 2020; accepted April 21, 2020 (Edited by M. Mizutani) Abstract Most leguminous plants produce (−)-type enantiomers of pterocarpans as the phytoalexin, but pea (Pisum sativum L.) produces the opposite stereoisomer of pterocarpan, (+)-pisatin. Biosynthesis of (−)-pterocarpan skeleton is completely characterized at the molecular level, and pterocarpan synthase (PTS), a dirigent (DIR) domain-containing protein, participates in the last dehydration reaction. Similarly, isoflav-3-ene, a precursor of (+)-pisatin, is likely to be biosynthesized by the DIR-mediated dehydration reaction; however the biosynthesis is still unknown. In the present study, we screened PTS homologs based on RNA-sequence data from (+)-pisatin-producing pea seedlings and demonstrated that one of the candidates encodes isoflav-3-ene synthase (I3S). Real-time PCR analysis revealed that transcripts of I3S, in addition to other genes involved in the (+)-pisatin pathway, transiently accumulated in pea upon elicitation prior to the maximum accumulation of (+)-pisatin. I3S orthologs were also found in soybean and Lotus japonicus that are not known to accumulate (+)-pterocarpan, and the catalytic function of gene products was verified to be I3S by the in vitro enzyme assay. Incubation of the crude extract of elicited soybean cells with isoflav-3-ene yielded coumestrol, suggesting that isoflav-3-ene is a precursor of coumestrol biosynthesis in soybean. Key words: coumestrol, isoflav-3-ene, phytoalexin, (+)-pisatin, Pisum sativum. (−)-type enantiomers, and only a limited number Introduction of plant species, such as peanut (Arachis hypogaea), Phytoalexins are plant-producing antimicrobial Japanese pagoda tree (Styphnolobium japonicum), and compounds induced by both biotic and abiotic pea (Pisum sativum), produce (+)-pterocarpans (Ingham stresses, and leguminous plants mainly produce 1979; Strange et al. 1985; VanEtten et al. 1989). The pterocarpan-based isoflavonoids as phytoalexins (Aoki stereochemistry of pterocarpan is important because it et al. 2000; Ingham 1982). Pterocarpans contain two determines the antimicrobial activity against pathogens; asymmetric carbons at C-6a and C-11a, but only two that is, some plant pathogens can detoxify (−)-isomers cis configurations are sterically possible and found but not (+)-isomers, and as a result, (+)-pterocarpans in nature (Figure 1) (Dewick 1986). All levorotatory show higher activity than the (−)-isomers (Delserone et pterocarpans are widely accepted to have the (6aR,11aR) al. 1992). The entire biosynthesis of (−)-pterocarpan was configuration, such as (−)-medicarpin (6a) and revealed by identifying pterocarpan synthase (PTS) as (−)-maackiain (6b), and dextrorotatory pterocarpans the long-standing missing link (Uchida et al. 2017), but have the opposite configuration, such as (+)-maackiain further studies are required to elucidate the biosynthesis (7a) and (+)-pisatin (7c) (Slade et al. 2005). Among the of (+)-pterocarpan. two enantiomers of pterocarpans, most legumes produce (+)-Pisatin (7c), which was the first chemically Abbreviations: DIR, dirigent; DMDI, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol; DMDIF, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene; DMI, 7,2′-Dihydroxy-4′-methoxyisoflavanol; DMIF, 7,2′-dihydroxy-4′-methoxyisoflav-3-ene; HPLC, high performance liquid chromatography; I2′H, isoflavone 2′-hydroxylase; I3S, isoflav-3-ene synthase; I4R, 2′-hydroxyisoflavanone 4-reductase; IFR, isoflavone reductase; LB, Luria Broth; NMR, nuclear magnetic resonance; PTS, pterocarpan synthase; RPKM, reads per kilobase of exon model per million mapped reads; RT-PCR, reverse transcription polymerase chain reaction; SOR, sophorol reductase; THIF, 7,2′,4′-trihydroxyisoflav-3-ene; TLC, thin-layer chromatography; VR, vestitone reductase. a Deceased This article can be found at http://www.jspcmb.jp/ Published online July 30, 2020 Copyright © 2020 Japanese Society for Plant Biotechnology 302 Isoflav-3-ene synthase of Pisum sativum Figure 1. Biosynthesis of (+)-pisatin and related compounds. Names of skeletons and individual compounds are shown in bold and plain fonts, respectively. Isoflav-3-enes are shown inside the dotted-line frame. Biosynthesis of isoflavones and 2′-hydroxyisoflavones shown here constitutes a metabolic grid, and enzymes involved are shown in parentheses. Constituents of isoflavonoids of leguminous plants are as follows: (6a) Glycyrrhiza spp. and Medicago spp., (6b) Maackia spp., Cicer spp., and Trifollium spp., (7a) Styphnolobium japonicum, (7c) Pisum sativum, (8) Glycine max, Glycyrrhiza spp., Lotus japonicus, and Medicago spp., (9) Cicer spp., (10) Lespedeza homoloba, and (11) Vigna unguiculate. Abbreviations: IFS, 2-hydroxyisoflavanone synthase; HID, 2-hydroxyisoflavanone dehydratase; HI4′OMT, 2-hydroxyisoflavanone 4′-O- methyltransferase; I2′H, isoflavone 2′-hydroxylase; I3′H, isoflavone 3′-hydroxylase; I3S, isoflav-3-ene synthase; IFR, isoflavone reductase; I4R, 2′-hydroxyisoflavanone 4-reductase; PBS, pseudobaptigenin synthase; PTS, pterocarpan synthase; SOR, sophorol reductase; VR, vestitone reductase; 2′HF, 2′-hydroxyformononetin; DMD, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavone; DMI, 7,2′-dihydroxy-4′-methoxyisoflavanol; DMDI, 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol; DMIF, 7,2′-dihydroxy-4′-methoxyisoflav-3-ene; DMDIF, 7,2′-dihydroxy-4′,5′- methylenedioxyisoflav-3-ene; THIF, 7,2′,4′-trihydroxyisoflav-3-ene. identified phytoalexin and is exclusively produced by pea hydroxyisoflavanones (Fischer et al. 1990; Paiva et al. (Cruickshank and Perrin 1960), is one of the best-studied 1991; Tiemann et al. 1991; Uchida et al. 2017). An IFR (+)-pterocarpans but its biosynthesis has only been isolated from (+)-pisatin-producing tissue of pea did partially elucidated. The biosynthesis of (−)-pterocarpan not produce the expected (3S)-2′-hydroxyisoflavanone has been found to involve two stereospecific and yielded only (3R)-enantiomer (Paiva et al. 1994). intermediates, namely (3R)-2′-hydroxyisoflavanone The involvement of an epimerase, which converts (3R)- and (3R,4R)-2′-hydroxyisoflavanol, the latter of sophorol (3b) into (3S)-sophorol, has been previously which is converted to (−)-pterocarpan by PTS (Figure suggested (Dewick 1986; Paiva et al. 1994). However, in 1). Thus, clarifying the branching point to produce a tracer experiment, 3H-labeled (3R)-sophorol (3b) was opposite stereoisomers is crucial for the elucidation efficiently incorporated into+ ( )-pisatin (7c) compared of (+)-pterocarpan biosynthesis. As isoflavone with that from (3S)-sophorol (DiCenzo and VanEtten reductase (IFR) catalyzes the first introduction step of 2006), showing no evidence to support the hypothetical chirality in pterocarpan biosynthesis, it was originally epimerase. postulated to be the pivotal enzyme that determines the The next step to IFR is the conversion of (3R)-2′- stereochemistry of the end product (Banks and Dewick hydroxyisoflavanone to (3R,4R)-2′-hydroxyisoflavanol, 1982a, b). Indeed, several IFRs of the (−)-pterocarpan- and 2′-hydroxyisoflavanone 4-reductase (I4R) mediates producing legumes, such as soybean (Glycine max), this reaction. I4R is also designated as a sophorol alfalfa (Medicago sativa), and chickpea (Cicer arietinum), reductase (SOR) in pea (DiCenzo and VanEtten converted achiral 2′-hydroxyisoflavones into (3R)-2′- 2006) and vestitone reductase (VR) in alfalfa (Guo Copyright © 2020 Japanese Society for Plant Biotechnology K. Uchida et al. 303 and Paiva 1995), and pea SOR specifically converts efficient synthesis of isoflav-3-enes from isoflavones (3R)-sophorol (3b) to (3R,4R)-7,2′-dihydroxy-4′,5′- was developed using co-cultured E. coli cells expressing methylenedioxyisoflavanol (DMDI, 4b). The suppressed several biosynthetic genes. Moreover, metabolic expression of the IFR and SOR genes by RNA-mediated correlation between the consumption of isoflav-3-ene genetic interference in the hairy roots of pea led and production of coumestrol was also observed in the to a decrease in the (+)-pisatin (7c) accumulation in vitro assay using crude extract of elicited soybean (Kaimoyo and VanEtten 2008). Taken together, these cells. The results achieved herein will offer a new results indicate that (+)-pisatin (7c) is biosynthesized perspective for the elucidation of stereoisomer-specific via (3R)-sophorol (3b) and (3R,4R)-DMDI (4b). (+)-pterocarpan biosynthesis, mediated by DIR domain- More recently, in vitro enzyme assays using a cell-free containing proteins that produce isoflav-3-ene. extract of (+)-pisatin-producing pea seedlings have demonstrated the conversion of (3R,4R)-DMDI (4b) Materials and methods to 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene (DMDIF, 5b). Consequently, the achiral isoflav-3- Chemicals ene was proposed to be an intermediate of (+)-pisatin Formononetin (1a) and coumestrol (8) were purchased from biosynthesis (Celoy and VanEtten 2014). Thus, the Tokyo Chemical Industry (Tokyo, Japan) and Sigma-Aldrich
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