Purification, Cloning, and Properties of an Acyltransferase Controlling Shikimate and Quinate Ester Intermediates in Phenylpropanoid Metabolism

• Published in: The Journal of biological chemistry Vol. 278; no. 1; pp. 95 - 103
• Main Authors: Hoffmann, Laurent, Maury, Stéphane, Martz, Françoise, Geoffroy, Pierrette, Legrand, Michel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03-01-2003; American Society for Biochemistry and Molecular Biology
• Subjects: Acyl Coenzyme A - metabolism; Acyltransferases - classification; Acyltransferases - genetics; Acyltransferases - isolation & purification; Acyltransferases - metabolism; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Coenzyme A - chemistry; Coenzyme A - metabolism; Dianthus - genetics; DNA, Plant - genetics; DNA, Plant - metabolism; Esters - chemistry; Esters - metabolism; Genes, Plant; Lignin - metabolism; Molecular Sequence Data; Molecular Structure; Nicotiana - chemistry; Nicotiana - genetics; Nicotiana - metabolism; Phenols - metabolism; Phenylalanine - metabolism; Phylogeny; Plant Structures - chemistry; Plant Structures - metabolism; Quinic Acid - chemistry; Quinic Acid - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Sequence Alignment; Shikimic Acid - chemistry; Shikimic Acid - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209362200

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show thatp-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.