A Structural Basis for the Biosynthesis of the Major Chlorogenic Acids Found in Coffee1[W][OA]

• Published in: Plant physiology (Bethesda) 2012-07, Vol.160 (1), p.249-260
• Main Authors: Lallemand, Laura A., Zubieta, Chloe, Lee, Soon Goo, Wang, Yechun, Acajjaoui, Samira, Timmins, Joanna, McSweeney, Sean, Jez, Joseph M., McCarthy, James G., McCarthy, Andrew A.
• Format: Article
• Language: English
• Subjects: Biochemical Processes and Macromolecular Structures
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.112.202051

Chlorogenic acids ( CGAs ) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee ( Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5- O -dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters ( CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.