TAILIEUCHUNG - Báo cáo khoa học: Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation ofcinnamicacid to formprecursorsofligninandmanyother phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to iden-tify active site residues likely togovern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directedmutagenesis. Active site modelling predicted that N302 and I371 form a hydro-gen bond and hydrophobic contacts with the anionic site or aromatic ringofthe substrate | Eur. J. Biochem. 270 3684-3695 2003 FEBS 2003 doi Key substrate recognition residues in the active site of a plant cytochrome P450 CYP73A1 Homology model guided site-directed mutagenesis Guillaume A. Schoch1 Roger Attias2 Monique Le Ret1 and Daniele Werck-Reichhart1 1 Department of Plant Stress Response Institute of Plant Molecular Biology Université Louis Pasteur Strasbourg France 2Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques Universite Paris V 45 Paris France CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to fomi precursors of linnin add mnno othrr phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modi i íication e t thne resid uss led to a drastic decrease in substrate binding and metabolism without major perturbation of protein strtucrlLre . Changes to residue K484 which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme did not influence initial substrate binding. However the K484M substitution led to a 50 loss in catalytic activity. K484 may affect positioning of the uuljttriite m the reduced enzyme during the catalytic cycle or product release. Catalytic analysis ofthe mutants with structural analogues of cinnamíc acid. in partícuair i ndoee-d-carboxylÍC acid that can be hydroxylated with different regioselectivi-ties supports the involvement of N302 1371 and K484 m substrate docking and orientation. Keywords active site cinnamate 4-hydroxylase homology modeling plant .

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