Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant—Ruta graveolens—exhibiting low sensitivity to psoralen inactivation q Antoine Gravot, a Romain Larbat, a Alain Hehn, a Karine Li  evre, a Eric Gontier, a Jean-Louis Goergen, b and Fr  ed eric Bourgaud a, * a UMR 1121 Agronomie Environnement INPL-INRA, ENSAIA 2, av. de la For^ et de Haye, 54505 Vandoeuvre-l es-Nancy Cedex, France b UPR-CNRS 6811 Laboratoire des Sciences du G enie Chimique, ENSAIA 2, av. de la For^ et de Haye, 54505 Vandoeuvre-l es-Nancy Cedex, France Received 23 September 2003, and in revised form 10 December 2003 Abstract Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 ( 0.2) for CYP73A1 and 1.1 ( 0.2) for CYP73A32. SDS–PAGE analysis demonstrated that [ 3 H]psoralen was irreversibly bound to the C4H apoprotein. K i and k inact for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenyl- propanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism. Ó 2003 Published by Elsevier Inc. Keywords: Cinnamic acid 4-hydroxylase; Furocoumarin; Mechanism-based inactivation; P450; Psoralen; Ruta Ruta graveolens is a Mediterranean medicinal plant producing high levels of linear furocoumarins, mostly psoralen and methoxypsoralens (Fig. 1). These mole- cules display the same basic structure, consisting of a furan ring condensed on a coumarin core. They also exhibit strong photoreactive properties and are com- monly used in dermatology for the treatment of psori- asis and vitiligo [1]. R. graveolens could present an alternative source of furocoumarins for the pharma- ceutical industry [2]. Many enzymes involved in the furocoumarin bio- synthesis belong to the cytochrome P450 superfamily [3,4]. Among them, cinnamate 4-hydroxylase (C4H) 1 is the first P450 upstream in the phenylpropanoid pathway leading to the synthesis of a variety of compounds in- cluding lignin monomers, flavonoids, and furocouma- rins. C4H catalyses the hydroxylation of cinnamic acid into coumaric acid and has been one of the most studied P450s in plant. Forty-one cDNAs coding for C4H have been cloned so far and are reported as CYP73 in the q GenBank Accession Nos: AF548370 (CYP73A32v2 cDNA) and AAN63028 (CYP73A32v2 protein). * Corresponding author. Fax: +33-383-59-57-99. E-mail address: bourgaud@ensaia.inpl-nancy.fr (F. Bourgaud). 1 Abbreviations used: C4H, cinnamic acid 4-hydroxylase; MBI, mechanism-based inactivator; 5-MOP, 5-methoxypsoralen; 8-MOP, 8- methoxypsoralen; 5,8-MOP, 5,8-dimethoxypsoralen; PAL, phenylala- nine ammonia-lyase; PIP, piperonilic acid. 0003-9861/$ - see front matter Ó 2003 Published by Elsevier Inc. doi:10.1016/j.abb.2003.12.013 Archives of Biochemistry and Biophysics 422 (2004) 71–80 ABB www.elsevier.com/locate/yabbi