Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis

Romain Larbat (1) , Sandra Kellner (2) , Silvia Specker (2) , Alain Hehn (1) , Eric Gontier (1) , Joachim Hans (2) , Frédéric Bourgaud (3) , Ulrich Matern

1 LAE - Laboratoire Agronomie et Environnement
2 Philipps Universität Marburg = Philipps University of Marburg
3 INPL - Institut National Polytechnique de Lorraine

Ammi majus L. accumulates linear furanocoumarins by cyto-chrome P450 (CYP)-dependent conversion of 6-prenylumbellif-erone via ()-marmesin to psoralen. Relevant activities, i.e. pso-ralen synthase, are induced rapidly from negligible background levels upon elicitation of A. majus cultures with transient maxima at 9 –10 h and were recovered in labile microsomes. Expressed sequence tags were cloned from elicited Ammi cells by a nested DD-RT-PCR strategy with CYP-specific primers, and full-size cDNAs were generated from those fragments correlated in abundance with the induction profile of furanocou-marin-specific activities. One of these cDNAs representing a transcript of maximal abundance at 4 h of elicitation was assigned CYP71AJ1. Functional expression in Escherichia coli or yeast cells initially failed but was accomplished eventually in yeast cells after swapping the N-terminal membrane anchor domain with that of CYP73A1. The recombinant enzyme was identified as psoralen synthase with narrow substrate specificity for ()-marmesin. Psoralen synthase catalyzes a unique carbon-chain cleavage reaction concomitantly releasing acetone by syn-elimination. Related plants, i.e. Heracleum mantegazzianum, are known to produce both linear and angular furanocoumarins by analogous conversion of 8-prenylumbelliferone via ()-columbianetin to angelicin, and it was suggested that angelicin synthase has evolved from psoralen synthase. However, ()-columbianetin failed as sub-strate but competitively inhibited psoralen synthase activity. Analogy modeling and docked solutions defined the conditions for high affinity substrate binding and predicted the minimal requirements to accommodate ()-columbianetin in the active site cavity. The studies suggested that several point mutations are necessary to pave the road toward angelicin synthase evolution.

Mots clés: ESCHERICHIA COLI

Mots clés: AMMI MAJUS

Domaines: Biologie moléculaire; Biochimie [q-bio.BM]; Botanique

Romain Larbat, Sandra Kellner, Silvia Specker, Alain Hehn, Eric Gontier, et al.. Molecular Cloning and Functional Characterization of Psoralen Synthase, the First Committed Monooxygenase of Furanocoumarin Biosynthesis. Journal of Biological Chemistry, 2007, 282 (1), pp.542-554. ⟨10.1074/jbc.M604762200⟩. ⟨hal-01738059⟩