
Purification, crystallization and preliminary X‐ray diffraction analysis of a hydroxycinnamoyl‐CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) from Coffea canephora involved in chlorogenic acid biosynthesis
Author(s) -
Lallemand Laura A.,
McCarthy James G.,
McSweeney Sean,
McCarthy Andrew A.
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112019082
Subject(s) - biosynthesis , shikimate pathway , crystallization , chemistry , o methyltransferase , coffea canephora , stereochemistry , biochemistry , enzyme , biology , methyltransferase , organic chemistry , methylation , gene , botany , coffea arabica
Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, including Coffea canephora (robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl‐CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl‐CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT from C. canephora in Escherichia coli as well as its purification and crystallization are presented. Crystals were obtained by the sitting‐drop technique at 293 K and X‐ray diffraction data were collected on the microfocus beamline ID23‐2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space group P 4 2 2 1 2 with unit‐cell parameters a = b = 116.1, c = 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl‐CoA‐dependent BAHD acyltransferase superfamily.