bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 Parallel evolution of UbiA superfamily proteins into aromatic O- 3 prenyltransferases in plants 4 5 Authors 6 Ryosuke Munakata1,2, Alexandre Olry2, Tomoya Takemura1, Kanade Tatsumi1, Takuji Ichino1, Cloé 7 Villard2, Joji Kageyama1, Tetsuya Kurata3, Masaru Nakayasu1, Florence Jacob4, Takao Koeduka5 8 Hirobumi Yamamoto6, Eiko Moriyoshi1, Tetsuya Matsukawa7,8, Jeremy Grosjean2, Célia Krieger2 1 9 10 2* 1* 9 Akifumi Sugiyama , Masaharu Mizutani , Frédéric Bourgaud , Alain Hehn , and Kazufumi Yazaki 10 11 *Author for correspondence 12 Kazufumi Yazaki 13 Tel: +81 774 38 3621 14 Email: [email protected] 15 16 Alain Hehn 17 Tel: +33 3 72 74 40 77 18 Email: [email protected] 19 20 Affiliation 21 1 Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto 22 University, Uji, Kyoto, 611–0011, Japan 23 2 Université de Lorraine, INRA, LAE, F54000, Nancy, France 24 3 EditForce Inc., Fukuoka 810-0001, Japan bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 4 PalmElit SAS, Montferrier sur Lez 34980, France 26 5 Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 27 753-8515, Japan 28 6 Department of Applied Biosciences, Faculty of Life Sciences, Toyo University, Izumino 1–1-1, 29 Itakura-machi, Ora-gun, Gunma 374-0193, Japan 30 7 The Experimental Farm, Kindai University, Wakayama, Japan 31 8 Faculty of Biology-Oriented Science and Technology, Kindai University, 32 Wakayama, Japan 33 9 Functional Phytochemistry, Graduate School of Agricultural Science, Kobe University, Kobe, 34 Japan. 35 10 Plant Advanced Technologies – PAT, 19 Avenue de la forêt de Haye, 54500 Vandoeuvre, France 36 37 ORCID 38 Ryosuke Munakata: 0000-0002-7888-6281 39 Alexandre Olry: 0000-0002-2008-9845 40 Kanade Tatsumi : 0000-0002-9810-9367 41 Takuji Ichino : 0000-0001-9058-5660 42 Cloé Villard: 0000-0001-6683-8541 43 Tetsuya Kurata : 0000-0002-7918-3027 44 Masaru Nakayasu : 0000-0002-6980-7238 45 Florence Jacob: 0000-0002-0454-1037 46 Takao Koeduka: 0000-0002-0786-3242 47 Hirobumi Yamamoto: 0000-0002-4958-4698 48 Eiko Moriyoshi: 0000-0002-8339-3529 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 Tetsuya Matsukawa: 0000-0003-3495-9634 50 Jeremy Grosjean: 0000-0002-1972-2180 51 Akifumi Sugiyama: 0000-0002-9643-6639 52 Masaharu Mizutani : 0000-0002-4321-0644 53 Frédéric Bourgaud: 0000-0002-9898-2625 54 Alain Hehn: 0000-0003-4507-8031 55 Kazufumi Yazaki: 0000-0003-2523-6418 56 57 Abstract 58 Plants produce approximately 300 aromatic molecules enzymatically linked to prenyl side 59 chains via C-O bonds. These O-prenylated aromatics have been found in taxonomically distant plant 60 taxa as compounds beneficial or detrimental to human health, with O-prenyl moieties often playing 61 crucial roles in their biological activities. To date, however, no plant gene encoding an aromatic O- 62 prenyltransferase (O-PT) has been described. This study describes the isolation of an aromatic O-PT 63 gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This 64 gene is responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug 65 pharmacokinetics in the human body. Another coumarin O-PT gene of the same protein family was 66 identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O- 67 prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation 68 activity evolved independently from the same ancestral gene in these distant plant taxa. These findings 69 shed light on understanding the evolution of plant secondary metabolites via the UbiA superfamily. 70 71 Introduction 72 Plants produce many O-prenylated aromatic molecules possessing prenyl side chains bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 attached to the aromatic cores via C-O bonds. These aromatic core structures include flavonoids, 74 coumarins, xanthones, and aromatic alkaloids, with roughly half of them (ca. 150 structures) being 75 classified as coumarins1,2. Some O-prenylated aromatics have pharmaceutical activities, whereas 76 others are deleterious to human health1,2. These beneficial/detrimental activities are often due to or 77 enhanced by O-prenyl moieties3–6. 78 Native coumarin O-prenyltransferases (O-PT) of Rutaceae and Apiaceae, plants that 79 accumulate large amounts of O-prenylated coumarins, have been characterized biochemically, with 80 members of the membrane-bound UbiA superfamily of proteins found to be involved in coumarin O- 81 prenylation7,8. To date, approximately 50 UbiA superfamily genes have been found to encode aromatic 82 C-PTs, which transfer prenyl moieties to aromatic cores via C-C bonds. Although these genes were 83 shown to encode enzymes involved in plant primary and secondary metabolism, no gene encoding an 84 aromatic O-PT has yet been identified in plants. O-Prenylated aromatic compounds have been detected 85 in several distant plant families, including Asteraceae, Boraginaceae, Fabaceae, Hypericaceae, 86 Rutaceae and Apiaceae, but are not ubiquitous throughout the plant kingdom1. The lack of knowledge 87 of aromatic O-PT genes has prevented a determination of the appearance of aromatic O-prenylation 88 activity during plant speciation. 89 Among Rutaceae, Citrus species accumulate large amounts of O-prenylated coumarins, 90 especially in their flavedo (outer pericarp)9–12. Citrus O-prenylated coumarins have shown various 91 pharmaceutical properties2, including anti-cancer4,13, anti-microbial3, and anti-inflammatory14 92 activities, although some of these derivatives have shown undesirable effects in humans. Citrus fruits 93 and juices enhance the bioavailability of orally administrated medications, which can lead to overdoses 94 and increased side effects5,15. The ‘grapefruit-drug interactions’ have been found to alter the 95 pharmacokinetics of more than 85 medications, including statins and calcium channel blockers15. The 96 United States Food and Drug Administration has cautioned consumers not to consume grapefruits or bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 grapefruit juice at times close to taking such drugs16. Citrus species are thought to alter drug 98 pharmacokinetics by inactivating CYP3A4, the major xenobiotic-metabolizing enzyme in the 99 intestines and liver5,15. 100 Furanocoumarins (FCs) are tricyclic coumarins containing a furan ring. O-geranylated 101 forms of FCs including bergamottin and its oxidative derivatives (Fig. 1a) are promising candidates 102 causing grapefruit-drug interactions due to their potent inhibition of CYP3A45,15,17. CYP3A4- 103 catalyzed metabolism of their furan rings produces reactive chemicals that inactivate this enzyme 104 itself18, while their O-geranyl side chains contribute to binding to the active site of CYP3A46 toward 105 metabolization of the furan ring. Bergamottin and 6’,7’-dihydroxybergamottin show 7- and 160-fold 106 higher in vitro inhibitory activity, respectively, than the non-geranylated form, bergaptol5. Furthermore, 107 O-geranyl moieties act as linkers to form FC dimers, called paradisins, which are more potent CYP3A4 108 inactivators than monomeric O-geranylated FCs5. These findings suggest that paradisins, along with 109 O-geranylated FC monomers, may be involved in grapefruit-drug interactions. 110 Starting with transcriptome analysis of flavedo tissues, this study describes the isolation of 111 a gene encoding a coumarin O-PT involved in bergamottin biosynthesis in grapefruit and the 112 functional characterization of its gene product. The contribution of O-PT orthologs to coumarin 113 biosynthesis was assessed in various Citrus species. In addition, an aromatic O-PT was isolated from 114 Angelica keiskei, an apiaceous medicinal plant producing O-prenylated coumarins19. The evolutionary 115 development of aromatic O-prenylation activity in plants was assessed by phylogenetic analysis of O- 116 PTs from taxonomically distant families Rutaceae and Apiaceae. 117 118 Results 119 Construction of a transcriptome dataset from grapefruit flavedo tissues 120 Because the native enzymes catalyzing coumarin O-prenylation in lemon flavedo were bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.192757; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 121 shown