A New Type of Peroxisomal Acyl-Coenzyme A Synthetase from Arabidopsis thaliana Has the Catalytic Capacity to Activate Biosynthetic Precursors of Jasmonic Acid

Claus Wasternack,Otto Miersch,Lucie Kienow,Elmon Schmelzer,Michael Bartsch,Thomas Colby,Erich Kombrink,Hans-Peter Stuible,Katja Schneider

doi:10.1074/jbc.m413578200

Abstract

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2'-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.

Highlights

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function
It was shown that one enzyme of this group, called JAR1, functions as a jasmonic acid (JA)-amino acid synthetase conjugating activated JA with isoleucine, and genetic evidence supports the notion that JA-Ile is an essential component of jasmonate signaling in Arabidopsis [5]
Large Scale in Vitro Substrate Analysis of Adenylate-forming Enzymes—In order to identify in vitro substrates of 4CL-like proteins and other carboxylic acid-activating enzymes operating via an adenylate intermediate, we established a sensitive enzyme assay, which allows the analysis of a large number of different compounds in parallel

Summary

EXPERIMENTAL PROCEDURES

Isolation and Cloning of At4g05160 and At5g63380 cDNAs—The cDNAs of At4g05160 and At5g63380 were amplified by RT-PCR from A. thaliana (Col-0) total RNA. The standard reaction mixture contained 2 ␮g of purified protein, 200 ␮M carboxylic acid substrate, 50 ␮M ATP, 250 ␮M MgCl2, 100 ␮M CoA, 1 mM dithioerythritol, and 0.1 M Tris-HCl (pH 7.5) in a total volume of 200 ␮l. Lipophilic substrates, such as longchain fatty acids, were dissolved in buffer containing 2% Triton X-100, leading to a final concentration of 0.1% Triton X-100 in the assay. Total RNA was isolated from 300 mg of cells using the RNeasy plant mini kit (Qiagen, Hilden, Germany), denatured, separated on 1.2% agarose-formaldehyde gels, transferred to nylon membranes (Biodyne B; Pall Life Sciences, New York), and hybridized with radiolabeled DNA probes as described previously [43]. All compounds represent mixtures of stereoisomers, which were used without further separation

RESULTS

35 Ϯ 10 11 Ϯ 5

DISCUSSION