Design of Fluorescent Substrates and Potent Inhibitors of CYP73As, P450s That Catalyze 4-Hydroxylation of Cinnamic Acid in Higher Plants † Michel Schalk, ‡ Yannick Batard, ‡ Andre ´ Seyer, § Svetlana Nedelkina, | Francis Durst, ‡ and Danie `le Werck-Reichhart* ,‡ De ´ partement d’Enzymologie Cellulaire et Mole ´ culaire, Institut de Biologie Mole ´ culaire des Plantes, Centre National de la Recherche Scientifique UPR 406, 28 rue Goethe, 67000 Strasbourg, France, Laboratoire de Chimie Bio-organique, Faculte ´ de Pharmacie, Centre National de la Recherche Scientifique URA 1386, 74 route du Rhin, 67048 Strasbourg Cedex, France, and Institute of Cytology and Genetics, 630090 NoVosibirsk-90, Russia ReceiVed June 30, 1997; ReVised Manuscript ReceiVed September 29, 1997 X ABSTRACT: CYP73As are the major functional cytochromes P450 in higher plants. Several of them have been shown to catalyze the 4-hydroxylation of cinnamic acid, the first oxidative step in the synthesis of lignin, flavonoids, coumarins, and other phenylpropanoids. The coding sequence for CYP73A1, the enzyme from Helianthus tuberosus, has been isolated and expressed in yeast. Previous studies indicate that the yeast-expressed enzyme is capable of metabolizing cinnamic acid and several small, planar molecules but with low efficiency. Using this we further examined how CYP73A1 could bind and metabolize a set of possible alternate substrates. We show here that naphthalenes, quinolines, and indoles substituted with an aldehyde, a carboxylic, or a sulfonic acid group make good ligands and substrates for CYP73A1. The best ligands are hydroxynaphthoic acids, which show higher affinity than cinnamate. Naphthalene, 2-naphthol, and molecules with two-carbon side chains, such as natural and synthetic auxins, are not substrates of this enzyme. Methyl-2-naphthoate and 2-hydroxy-1-naphthoic acid are strong ligands of CYP73A1 but are not metabolized. Uncoupling and low spin conversion induced by these compounds suggest that their positioning in the heme pocket is inadequate for catalysis. These compounds can act as potent inhibitors of the second step of the phenylpropanoid pathway, the first described so far. The molecule which most closely mimics cinnamic acid, 2-naphthoic acid, is metabolized with a catalytic turnover and efficiency similar to those measured with the physiological substrate. Using this compound we designed a fluorometric assay to measure the catalytic activity of CYP73As. This assay was then used to monitor the CYP73As activity in microsomes from transgenic yeast and several plant species. Cytochromes P450 from the CYP73A subfamily catalyze the 4-hydroxylation of cinnamic acid. This oxygenation reaction constitutes the second step in the plant-specific pathway of phenylpropanoid metabolism, that leads to the synthesis of lignin, anti-UV and insect attracting pigments, and several classes of defense-related molecules (1). The genes of at least 15 members of this P450 subfamily have now been isolated from different sources, including herba- ceous and woody monocot and dicot plants. Theses genes usually share more than 85% identity at the amino acid level and possess long stretches of totally conserved sequences. Interest in these P450s rests with their position upstream of the major pathway of plant secondary metabolism and its possible control. Better understanding of the roles of these enzymes holds significant physiological and economic importance. This is true with regard to lignin synthesis and pathogen resistance. The quantitative abundance of this enzyme in some plants suggests a crucial role, where CYP73A1 can represent up to 56% of the total P450 in wounded plant tissues (2). This may also be crucial for plants ability to process a significant proportion of their biomass, as lignin represents up to 35% of the dry weight of some woody plants (3). Finally, the possible control of the flux of metabolites between the main branch of the pathway and the synthesis of C6-C1 compounds, includes the biosynthesis of salicylic acid, a crucial signaling molecule that has been implicated in flowering, thermogenesis, and disease resistance (4). As a consequence, cinnamate hy- droxylase may be an ideal target for manipulating lignifi- cation or defense-related reactions in plants. Moreover, if CYP73As are also capable of metabolizing xenobiotics, as suggested by previous studies, then their abundance in the plant kingdom may also confer resistance to environmental toxins. Plant are believed to act as metabolic sinks for environmentally harmful molecules (5). To assess these possible capabilities, we developed procedures to evaluate the metabolic capacity of these widespread and highly expressed oxygenases. CYP73A1 is the cinnamate 4-hydroxylase (C4H) 1 from Helianthus tuberosus, a member of the Asteraceae family, closely related to sunflower. The coding sequence of the enzyme isolated from tuber tissues (6) was expressed in yeast (7). In microsomes from transgenic yeast, CYP73A1 † This work was supported by a MRES grant to M.S. * Corresponding author. Fax: 33 3 88 35 84 84. E-mail: daniele.werck@ibmp-ulp.u-strasbg.fr. ‡ CNRS UPR 406. § CNRS URA 1386. | Institute of Cytology and Genetics. X Abstract published in AdVance ACS Abstracts, November 15, 1997. 1 Abbreviations: C4H, trans-cinnamate 4-hydroxylase [NADPH: oxygen oxidoreductase (4-hydroxylating), EC1.14.13.11]; 2-NA, 2-naph- thoic acid; NAH, 2-naphthoic acid 6-hydroxylase; HS, high spin; tR, retention time. 15253 Biochemistry 1997, 36, 15253-15261 S0006-2960(97)01575-4 CCC: $14.00 © 1997 American Chemical Society