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

Laurent Hoffmann,Stéphane Maury,Françoise Martz,Pierrette Geoffroy,Michel Legrand

doi:10.1074/jbc.m209362200

Abstract

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 that p-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.

Highlights

Phenylpropanoid compounds are derived from phenylalanine by the action of phenylalanine ammonia-lyase, the branch point enzyme between primary and secondary metabolism [1, 2]
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 4-hydroxycinnamoyl-CoA ligase catalyzes the formation of CoA esters of hydroxycinnamic acids, which are activated intermediates in the biosynthesis of diverse compounds via specific branches of the pathway leading to lignins, lignans, flavonoids and isoflavonoids, stilbenes, coumarins, and numerous esters and amides [6, 7]. 4-Hydroxycinnamoyl-CoA ligase from various plant species accepts p-coumarate, ferulate, and caffeate as substrates in this decreasing preferential order, but does not accept sinapate [8, 9]

Summary

EXPERIMENTAL PROCEDURES Chemicals and Enzymes

Used chemicals and reagents were of the highest purity readily available. Bradford protein dye reagent was purchased from Bio-Rad (Marnes-la-Coquette, France). Plasmid and PCR products were extracted and purified from agarose gels using kits purchased from QIAGEN Inc. The amplified DNA was resolved by agarose gel electrophoresis, and the band of the expected size (1130 bp) was isolated and subcloned into pCRII-TOPO (Invitrogen) prior to sequencing. Spectrophotometric Assay—During enzyme purification from plant extracts, 10 nmol of p-coumaroyl-CoA was added to 100 ␮l of protein mixture and incubated at 30 °C for 1 h. Standard Assay Conditions for the Recombinant Enzyme—The reaction mixture contained (in a total volume of 20 ␮l) 100 mM phosphate buffer (pH 6.6), 1 mM dithiothreitol, 20 ng to 1 ␮g of purified enzyme, and the different substrates at 10 ␮M to 10 mM.

RESULTS

DISCUSSION

50 Ϯ 20 350 Ϯ 100