Biochemical Characterization of Sinapoylglucose:Choline Sinapoyltransferase, a Serine Carboxypeptidase-like Protein That Functions as an Acyltransferase in Plant Secondary Metabolism

Amber M Shirley,Clint Chapple

doi:10.1074/jbc.m302362200

Abstract

Recently, serine carboxypeptidase-like (SCPL) proteins that catalyze transacylation reactions in plant secondary metabolism have been identified from wild tomato and Arabidopsis. These include sinapoylglucose: choline sinapoyltransferase (SCT), an enzyme that functions in Arabidopsis sinapate ester synthesis. SCT and the other known SCPL acyltransferases all share the conserved serine, aspartic acid, and histidine residues employed for catalysis by classical serine carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown. To characterize further SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the serine carboxypeptidase, carboxypeptidase Y, and other proteins normally targeted to the vacuole. When expressed in this strain, SCT is similarly secreted. SCT has been purified from the yeast medium and used for kinetic characterization of the protein. Immunological analysis of SCT has revealed that the expected 50-kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30- and 17-kDa polypeptide.

Highlights

In Arabidopsis and some other members of the Brassicaceae, the end products of the phenylpropanoid pathway include the sinapate esters, sinapoylmalate and sinapoylcholine
In light of the recent cloning of SCT from Arabidopsis and the identification of SCT as an serine carboxypeptidase-like (SCPL) protein [5], in this paper we describe a detailed analysis of activity, substrate specificity, and processing of SCT
Kinetic Analysis of SCT Activity—Unlike other methods previously used for the measurement of SCT activity [21], the HPLC method used in this study enabled the simultaneous detection of sinapoylcholine and any other sinapoylglucosederived products, including sinapic acid, a potential spontaneous or enzyme-catalyzed hydrolytic product

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains—The construction of the S. cerevisiae strain W2579 (MATa ⌬prc vpl leu leu112 ura3-52) has been described [22]. The cell lysate was centrifuged at 3,000 ϫ g for 10 min and assayed for SCT activity as described below. After 24 h, the cultures were centrifuged at 2,000 ϫ g for 10 min to pellet the cells, and the supernatant was assayed directly for CPY activity by monitoring the decrease in absorbance at 339 nm of 0.2 mM N-(3-[2-furyl]acryloyl)-Phe-Phe (Sigma) in 50 mM MES and 1 mM EDTA (pH 6.5) over a 30-min period, as described previously [28]. LC separations were performed using a PuresilTM C18 column (Waters) (120-Å pore size, 5-␮m particle size) with a 23-min gradient from 1.5% acetic acid to 70% acetonitrile at a flow rate of 1 ml minϪ1 using UV detection at 335 nm.

Characterization of SCT

RESULTS

Specific activity

DISCUSSION