Two novel hqt genes show different specificities in the pathway of chlorogenic acid synthesis in artichoke

Artichoke is an edible vegetable, used not only as a food, but also for its positive medical properties, mostly attributed to polyphenols, a class of compounds with beneficial effects on human health. Mono- and dicaffeoylquinic acids (e.g. chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g. luteolin-7-O-glucoside) are the main phenolic constituents of artichoke extracts, with chlorogenic acid (CGA) being the most abundant. In plants, the synthesis of this compound is supposed to occur following three possible metabolic routes, and the enzyme hydroxycynnamoyl-quinate transferase (HQT), belonging to the acyl transferase BAHD family, is reported to be involved in the last steps of this biochemical pathway. In the present contribution, we report on the isolation and characterization of two artichoke full-length hqt and hqt2 cDNA sequences. Genomic organization of the genes revealed that they contain two exons and one intron. The obtained sequences showed a high level of similarity to homologous genes from other plant species, particularly tobacco and tomato. The transcripts of the two artichoke genes were expressed in E. coli, and biochemical assays were performed, in order to assess specificity of the enzymes for various substrates. Results indicate that quinate is preferred over shikimate, confirming that the two artichoke HQT enzymes possess an activity similar to other HQTs, however the preferred acyl donor seems to be different for the two enzymes. Comparative models of artichoke HQT and HQT2 were built based upon the available crystallized structures of two BAHD enzymes, by using the computer application MODELLER. The structural properties of HQT and HQT2 3D models with the best energy function were evaluated by using the WHATIF biochemical/computational tools. Potential binding sites of HQT and HQT2 were predicted by means of docking analyses. AUTODOCK was employed to evaluate the fit of potential substrates within the proposed binding sites. Real time PCR of hqt and hqt2 from different plant parts and developmental stages revealed that the two genes are differentially expressed in artichoke plants. In conclusion, both hqt and hqt2 are implicated in the synthesis of CGA, but possibly at different steps of the metabolic pathway, and according to the plant exigencies.