Chlorogenic acids (CGAs) represent a family of esters formed between a cinnamic acid derivative and quinic or shikimic acid. CGAs are secondary metabolites produced via the phenylpropanoid pathway by higher plants and are a major source of dietary antioxidants. Hydroxycinnamoyl-CoA esters are the precursors for CGAs and other phenolic compounds such as lignins. These activated intermediates are synthesized from a hydroxycinnamic acid and coenzyme A by 4-coumarate CoA ligase (4CL), which belongs to the adenylate-forming enzyme superfamily. Nicotiana tabacum 4CL2 was used to produce hydroxycinnamoyl-CoA esters and its structure was solved by molecular replacement. Two genes encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases from Coffea canephora were cloned. CcHCT and CcHQT, which belong to the acyl-CoA-dependent acyltransferase superfamily, were overexpressed in E. coli and purified to homogeneity. X-ray diffraction analysis of CcHCT crystals resulted in a structural solution by molecular replacement. A homology model was derived for CcHQT in order to propose some determinants of the preference for quinic or shikimic acid. Docking experiments were carried out in order to identify potential residues involved in enzyme-substrate interactions. High performance liquid chromatography analysis of enzymatic reactions showed that these enzymes are capable of synthesizing 5-O-caffeoylquinic acid but also the diester 3,5-O-dicaffeoylquinic acid, which is a major component of the coffee grain before ripening. The production of variants by site-directed mutagenesis enabled the identification of residues important for catalysis of the mono- and diacyltransfer reactions. The combined approach of structural biology and enzymology provides molecular insights into the role of HCT and HQT in CGA biosynthesis.
Structural and biochemical characterisation of enzymes involved in chlorogenic acid biosynthesis