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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site¹

Department of Plant Stress Response, Institute of Plant Molecular Biology, Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 28 rue Goethe, F–67000 Strasbourg, France (G.A.S., D.W.-R.); Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université René Decartes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8601, 45 rue des Saints-Pères, F–75270 Paris cedex 06, France (R.A., P.M.D.); and Institut Européen de Chimie et Biologie-Ecole polytechnique, 16, avenue Pey Berland, F–33607 Pessac cedex, France (M.B.)

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L^–1 yeast culture. This water-soluble enzyme was suitable for ¹H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and ¹H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the ¹H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle.

¹ This work was supported by the Centre National de la Recherche Scientifique Programme Chimie-Physique du Vivant and by a fellowship from the French Ministry of Research to G.A.S.

^* Corresponding author; e-mail daniele.werck{at}ibmp-ulp.ustrasbg.fr; fax 33–3–90–24–18–84.

Received January 10, 2003; returned for revision June 1, 2003; accepted August 13, 2003.