CYP98A3 from Arabidopsis thaliana Is a 3′-Hydroxylase of Phenolic Esters, a Missing Link in the Phenylpropanoid Pathway

• Published in: The Journal of biological chemistry Vol. 276; no. 39; pp. 36566 - 36574
• Main Authors: Schoch, Guillaume, Goepfert, Simon, Morant, Marc, Hehn, Alain, Meyer, Denise, Ullmann, Pascaline, Werck-Reichhart, Danièle
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.09.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Arabidopsis - enzymology; Arabidopsis Proteins; Arabidopsis thaliana; Biochemistry; Biochemistry, Molecular Biology; Botanics; caffeic acid; Cell Line; chlorogenic acid; Chromatography, High Pressure Liquid; coumaric acid; Coumaric Acids - chemistry; Coumaric Acids - metabolism; Coumaric Acids - pharmacology; CYP98A3 gene; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; DNA, Complementary - metabolism; Evolution, Molecular; Free Radical Scavengers - chemistry; Free Radical Scavengers - metabolism; Free Radical Scavengers - pharmacology; Life Sciences; lignin; Lignin - biosynthesis; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Models, Chemical; Molecular biology; Phylogeny; Propionates; Protein Binding; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity; Time Factors; Tissue Distribution; Vegetal Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104047200

The 4- and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid fromp-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues. Recombinant CYP98A3 expressed in yeast did not metabolize freep-coumaric acid or its glucose or CoA esters,p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-d-quinate esters oftrans-p-coumaric acid into the corresponding caffeic acid conjugates. The shikimate ester was converted four times faster than the quinate derivative. Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root. Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers. This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis.