title: Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.
abstract: 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 mM, 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.
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, 38106 Braunschweig, Germany.
authors.forename: Mariam M
authors.initials: MM
authors.lastname: Gaid
authors.forename: Debabrata
authors.initials: D
authors.lastname: Sircar
authors.forename: Andreas
authors.initials: A
authors.lastname: Müller
authors.forename: Till
authors.initials: T
authors.lastname: Beuerle
authors.forename: Benye
authors.initials: B
authors.lastname: Liu
authors.forename: Ludger
authors.initials: L
authors.lastname: Ernst
authors.forename: Robert
authors.initials: R
authors.lastname: Hänsch
authors.forename: Ludger
authors.initials: L
authors.lastname: Beerhues
mesh_terms: D000595:Amino Acid Sequence; D001565:Benzoates / Q000737:chemistry / Q000378:metabolism*; D002412:Cations; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002934:Cinnamates / Q000378:metabolism*; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D020902:Hypericum / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D021381:Protein Transport; D016415:Sequence Alignment; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D044004:Xanthones / Q000737:chemistry / Q000378:metabolism*; D000094162:Phytoalexins
chemical_list: D001565:Benzoates; D002412:Cations; D002934:Cinnamates; D012717:Sesquiterpenes; D044004:Xanthones; C009689:xanthone; D010650:Phenylalanine Ammonia-Lyase; D003066:Coenzyme A Ligases; D000094162:Phytoalexins