title: Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis.
abstract: The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.
authors.affiliation: UMR 1121 Nancy Université-Institut National de la Recherche Agronomique Agronomie Environnement Nancy-Colmar, 2 Avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Larbat
authors.forename: Alain
authors.initials: A
authors.lastname: Hehn
authors.forename: Joachim
authors.initials: J
authors.lastname: Hans
authors.forename: Sarah
authors.initials: S
authors.lastname: Schneider
authors.forename: Hélène
authors.initials: H
authors.lastname: Jugdé
authors.forename: Bernd
authors.initials: B
authors.lastname: Schneider
authors.forename: Ulrich
authors.initials: U
authors.lastname: Matern
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bourgaud
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019143:Evolution, Molecular; D011564:Furocoumarins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D028529:Pastinaca / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D011564:Furocoumarins; D010940:Plant Proteins; C046733:columbianetin; D003577:Cytochrome P-450 Enzyme System; C011659:angelicin; C050853:psoralen synthase