Cinnamate:CoA Ligase Initiates the Biosynthesis of a Benzoate-Derived Xanthone Phytoalexin in Hypericutn calycinum Cell Cultures

• Published in: Plant physiology (Bethesda) Vol. 160; no. 3; pp. 1267 - 1280
• Main Authors: Gaid, Mariam M., Sircar, Debabrata, Müller, Andreas, Beuerle, Till, Liu, Benye, Ernst, Ludger, Hänsch, Robert, Beerhues, Ludger
• Format: Journal Article
• Language: English
• Published: American Society of Plant Biologists 01.11.2012
• Subjects: Amino acids; BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES; Biosynthesis; Cell culture techniques; Complementary DNA; Enzymes; Esters; Parabens; Plants; Polymerase chain reaction; Xanthones
• ISSN: 0032-0889; 1532-2548

Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate: CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 HIM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate: CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.