Here, we report the isolation of the cDNA of a novel acyl transferase involved in C. cardunculus PP biosynthesis, and assess its Enzalutamide side effects leaf expression level as induced by UV C irradiation. We also derive the map location of this gene, along Inhibitors,Modulators,Libraries with that of the HCT gene described by Comino et al. Results Isolation and cloning of a full length HQT cDNA of globe artichoke and cardoon CODEHOP was used to target conserved acyltrans ferases in globe artichoke, resulting in the amplification of an incomplete internal acyltrans ferase like sequence, which was extended in both globe artichoke and cultivated cardoon via a RACE strategy. Full length cDNA sequences have been deposited in Gen bank. The genes are of identical length and their translation product is a 444 residue peptide with a molecular mass of 50 kDa.
The best matches obtained from a local alignment search within a non redundant protein database were with a sweet potato HCBT, Inhibitors,Modulators,Libraries and a tobacco HQT, which belongs to a multifunctional superfamily of plant acyl transferases. The sequences contain a HTLSD peptide, as does the HCT iso lated by Comino et al. matching Inhibitors,Modulators,Libraries the highly con served Inhibitors,Modulators,Libraries HXXXD motif characteristic for acyl transfer proteins. The DFGWG block present in other acyl transferases of the BAHD family is present from aa 391 to 395. Phylogenetic analyses confirmed that the isolated sequence showed a high degree of similarity with other already isolated HQT sequences and with HCTs from globe artichoke, coffee, tobacco and Arabidopsis. Heterologous expression of globe artichoke HQT in E.
coli and enzyme assays The globe artichoke acyltransferase cDNA was cloned and expressed in E. coli, using a pET3a expression vector. SDS PAGE analysis demonstrated the presence of a heterolo gous protein of apparent molecular mass 50 kDa both in the supernatant and in the pellet frac tion. This protein was absent from the equivalent Inhibitors,Modulators,Libraries frac tions of cultures of bacteria carrying an empty pET3A plasmid. The recombinant enzyme was incubated in the presence of p coumaroyl CoA or caffeoyl CoA and quinic acid or shikimic acid as substrates, and the products of the reactions were analyzed by HPLC. In the presence of active enzyme, new products were detected in the reaction mixtures contain ing p coumaroyl CoA or caffeoyl CoA and quinic acid. These products could not be detected when reactions were performed with the control crude extract. No sig nificant peaks were detected after addition of shikimic acid instead of quinic acid in the reaction mixture. Each reaction selleck chemicals llc product was identified by comparing its retention time and absorbance spectrum with authentic samples or isolated compounds previously characterized.