Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis Romain Larbat, Alain Hehn, Joachim Hans, Sarah Schneider, Hélène Jugdé, Bernd Schneider, Ulrich Matern, Frédéric Bourgaud To cite this version: Romain Larbat, Alain Hehn, Joachim Hans, Sarah Schneider, Hélène Jugdé, et al.. Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, 284 (8), pp.4776-4785. 10.1074/jbc.M807351200. hal-01738063 HAL Id: hal-01738063 https://hal.archives-ouvertes.fr/hal-01738063 Submitted on 20 Mar 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 8, pp. 4776–4785, February 20, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis* Received for publication, September 23, 2008, and in revised form, December 19, 2008 Published, JBC Papers in Press, December 19, 2008, DOI 10.1074/jbc.M807351200 Romain Larbat‡1, Alain Hehn‡1, Joachim Hans§, Sarah Schneider§,He´le`ne Jugde´ ‡, Bernd Schneider¶, Ulrich Matern§, and Fre´de´ric Bourgaud‡2 From the ‡UMR 1121 Nancy Universite´-Institut National de la Recherche Agronomique Agronomie Environnement Nancy-Colmar, 2 Avenue de la Foreˆt de Haye, 54505 Vandoeuvre-le`s-Nancy, France, the §Institut fu¨r Pharmazeutische Biologie, Philipps-Universita¨t Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany, and the ¶Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-Strasse 8, 07745 Jena, Germany The biosynthesis of linear and angular furanocoumarins is ous other bioactivities have been attributed to furanocouma- Downloaded from still poorly understood at the molecular level, with only psoralen rins, which are the focus of many pharmacological screenings, synthase (CYP71AJ1) identified from Ammi majus. Using cDNA although their mutagenic potential was recognized many years probes inferred from CYP71AJ1, three orthologs were isolated ago (3, 4). Furanocoumarins are formed by only four plant fam- from Apium graveolens (CYP71AJ2) and Pastinaca sativa ilies (i.e. Apiaceae, Rutaceae, Fabaceae, and Moraceae) (5, 6). (CYP71AJ3 and -4) and functionally expressed in yeast cells. They are allelochemicals that have been assigned ecological CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, functions, such as the defense against phytopathogens, germi- www.jbc.org whereas CYP71AJ4 only catalyzed the conversion of (؉)-colum- nation inhibition of competitor plants, or herbivore deterrence bianetin to angelicin and negligible amounts of a hydroxylated (7–10). Their toxicity is based on their ability to create covalent columbianetin by-product. CYP71AJ4 thus constitutes the first adducts of double-stranded DNA, impeding DNA replication at Universite Louis Pasteur on February 24, 2009 fully characterized P450 monooxygenase specific for the angu- (9, 11), as well as to inhibit cytochrome P450 enzymes (12–14). lar furanocoumarin pathway. The angelicin synthase exhibited It is noteworthy that plants synthesize either linear furanocou- ␮ ؉ ؎ an apparent Km of 2.1 0.4 M for ( )-columbianetin and a kcat marins (psoralens) only or both linear and angular furanocou- ؊1 ؅ ؉ ؎ of 112 14 min . Moreover, the use of 3 -deuterated ( )- marins, which are less widely distributed and mainly confined columbianetin as substrate led to an almost complete “meta- to some Fabaceae and Apiaceae. The association of both forms ؅ ؅ bolic switch,” resulting in the synthesis of anti-3 -hydroxy-3 - was demonstrated to be more toxic for insect larvae than the ؉ deuterated( )-columbianetin. This confirms that angelicin presence of linear furanocoumarins alone, which could reflect synthase attacks columbianetin by syn-elimination of hydrogen the evolutionary adaptation of plants to new herbivores that ؅ from C-3 . Sequence comparison between psoralen synthase have acquired the capacity to detoxify psoralens (15–17). (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% Due to the complex bioactivity of furanocoumarins, its bio- identity, whereas the identity dropped to 40% in those synthesis has received continuous attention. The basis of linear regions thought to provide the substrate recognition sites. furanocoumarin formation was mostly established by the end Accordingly, CYP71AJ3 and CYP71AJ4 might be derived of the 1980s by a combination of precursor feeding experiments from a common ancestor of unknown functionality by gene and the biochemical characterization of major enzymes of the duplication and subsequent molecular evolution. pathway. Nevertheless, until recently, the pathway was thought to depend on the ortho-hydroxylation of trans-orcis-4-cou- maric acid and cyclization of the product to umbelliferone (see Furanocoumarins are natural plant metabolites character- Ref. 6 for a review). However, a dioxygenase has now been ized by a furane moiety fused to benzopyran-2-one. The posi- cloned from Arabidopsis and expressed; this enzyme converts tion of the furane substitution distinguishes two large groups of feruloyl-CoA to 2,4-dihydroxy-5-methoxycinnamoyl-CoA and compounds, the linear (psoralens) and the angular furanocou- scopoletin (18). To a lesser extent, it also accepts 4-coumaroyl- marins (angelicin and derivatives) (Fig. 1) (1, 2). The psoralens CoA, which yields umbelliferone (Fig. 1). The data prove for the in particular are known for their photosensitizing and photo- first time the essential role of CoA-esters. These serve as an toxic effects and have been used in photochemotherapy of skin active substrate in coumarin biosynthesis, whereas unconju- disorders (psoriasis, vitiligo, and mycosis). In addition, numer- gated cinnamic acids are inactive. This pivotal role of CoA- esters in coumarin synthesis is consistent with the report of a * This work was supported by Re´gion Lorraine (to F. B.), Deutsche Forsch- unique 4-coumaroyl-CoA ligase that was specifically assigned ungsgemeinschaft, and Fonds der Chemischen Industrie (to U. M.). The costs of publication of this article were defrayed in part by the payment of to coumarin synthesis in Ruta graveolens (19). Umbelliferone is page charges. This article must therefore be hereby marked “advertise- subsequently prenylated to demethylsuberosin by a 6-prenyl- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. transferase also assigned to plastids in R. graveolens (20). The 1 Both of these authors contributed equally to this work. 2 To whom correspondence should be addressed. Tel.: 33-383-59-58-64; Fax: conversion of demethylsuberosin to marmesin and further to 33-383-59-57-99; E-mail: [email protected]. psoralen is then catalyzed by two distinct P450 enzymes (i.e. 4776 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 8•FEBRUARY 20, 2009 P. sativa Angelicin Synthase marmesin synthase and psoralen synthase) in Ammi majus and CYP71AJ2) and Pastinaca sativa (parsnip CYP71AJ3). Addi- Petroselinum crispum (21–23). Psoralen is next hydroxylated at tionally, the closely related CYP71AJ4 was isolated from pars- C-5 by yet another P450 monooxygenase-generating bergaptol, nip and annotated as angelicin synthase, the first P450 gene which is subsequently O-methylated to bergapten (5-me- committed to angular furanocoumarin biosynthesis. Align- thoxypsoralen) (24, 25). Alternatively, the formation of xantho- ments of the paralogs (CYP71AJ3 and CYP71AJ4) revealed that toxin (8-methoxypsoralen) from psoralen is supposed to pro- the sequence identity dropped to only 40% in substrate recog- ceed via analogous reactions (i.e. successive hydroxylation at nition sites (SRSs)3 at an overall identity of 70%. Assays con- C-8 and O-methylation), but this remains to be established. The ducted with the recombinant enzyme and syn-[3Ј-D]columbi- pathway to angular furanocoumarins has not been as well doc- anetin demonstrated a reaction mechanism analogous to that umented, especially at the level of enzyme activity. This path- of psoralen synthase initiated by the abstraction of syn-C-3Ј- way branches off from that for psoralens by divergent prenyla- hydrogen. These results demonstrate that the CYP71AJ sub- tion of umbelliferone (Fig. 1) to form osthenol. The two family is involved in the synthesis of both linear and angular subsequent steps leading to angelicin were assumed to be very furanocoumarins and suggest that CYP71AJ3 and CYP71AJ4 similar to the steps leading to the conversion of demethylsub- derived from a common ancestor gene. erosin to (ϩ)-marmesin and psoralen. This assumption is par- ticularly convincing when the catalytic mechanisms of psoralen EXPERIMENTAL PROCEDURES synthase and angelicin synthase are compared. Previous studies Plant Material—Parsnip (Pastinaca sativa) plants