Molecular analysis of coenzyme A ligase from benzoate-metabolizing Sorbus aucuparia cell cultures

Benzoic acids are precursors and signal molecules. The objective of this work was to study benzoic acid biosynthesis at the molecular genetic level. In elicitortreated cell cultures of Hypericum androsaemum and Sorbus aucuparia, benzoic acid is a precursor of xanthone and biphenyl biosynthesis, respectively. Benzoic acid formation in H. androsaemum proceeds via cinnamic acid, with the underlying mechanism being CoA-dependent and non-ß-oxidative. At the biochemical level, three CoA ligases with different substrate specificities were detected. A full-length cDNA with homology to 4-coumarate:CoA ligase (4CL) was identified from yeast-extract-treated Sorbus aucuparia cell cultures using degenerate primers derived from conserved regions of 4CLs and gene specific primers for RTPCR. In addition, various partial CoA ligase cDNA sequences were cloned from both cell cultures. The full-length sequence contained a 1815 bp open reading frame (ORF) encoding a polypeptide of 605 amino acids. The encoded protein was successfully expressed in E. coli and purified by affinity chromatography. Most hydroxycinnamic acids were converted to the corresponding hydroxycinnamoyl-CoA thioesters, with 4-coumaric acid being the preferred substrate, followed by caffeic, m-coumaric, o-coumaric, ferulic, and cinnamic acids. Thus, the recombinant enzyme was a 4CL. No evidence was found for the conversion of sinapic acid to sinapoyl-CoA. Interestingly, S. aucuparia 4CL also exhibited activity toward benzoic acids. Benzoic, 2-hydroxybenzoic, and 3-hydroxybenzoic acids were activated at relative efficiencies of 12, 5, and 7%, respectively, compared to the activity with 4-coumaric acid. To our knowledge, this is the first plant 4CL showing distinct activity with benzoic acids. For the preferred substrate 4-coumaric acid, the Km and kcat/Km values were 9 ± 3 μM and 2.6 × 104 M-1sec-1, respectively. The Km values for CoA and ATP were 16 and 13 μM, respectively. Based on the amino acid sequences of 4CLs, a phylogenetic tree was constructed. S. aucparia 4CL belonged to the class II angiosperm 4CL sequences and grouped closely together with Rubus idaeus 4CL3.