Open AccessPurification, crystallization and preliminary X‐ray crystallographic analysis of hydroxycinnamoyl‐coenzyme A hydratase‐lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism
Author(s) -
Leonard Philip M.,
Marshall Caroline M.,
Dodson Eleanor J.,
Walton Nicholas J.,
Grogan Gideon
Publication year - 2004
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444904024588
Subject(s) - lyase , chemistry , phenylpropanoid , decarboxylation , coenzyme a , ferulic acid , stereochemistry , pseudomonas fluorescens , aldehyde , fumarase , enzyme , organic chemistry , biochemistry , biosynthesis , catalysis , biology , bacteria , reductase , genetics
4‐Hydroxycinnamoyl‐coenzyme A hydratase‐lyase (HCHL), also called feruloyl‐CoA hydratase‐lyase (FCHL), from Pseudomonas fluorescens strain AN103 is an enzyme of the crotonase superfamily that catalyses the one‐step conversion of the CoA thioesters of 4‐coumaric acid, caffeic acid and ferulic acid to the aromatic aldehydes 4‐hydroxybenzaldehyde, protocatechuic aldehyde and vanillin, respectively. The reaction occurs via a hydration followed by a carbon–carbon bond‐cleavage reaction. HCHL has been crystallized by the hanging‐drop method of vapour diffusion using polyethylene glycol 20 000 Da as the precipitant. The crystals belong to the orthorhombic system, with proposed space group P 2 1 2 1 2 and unit‐cell parameters a = 154.2, b = 167.5, c = 130.8 Å. The V M suggests that the asymmetric unit contains four trimers. Single‐wavelength data collection has been undertaken and structure determination is under way by molecular replacement using data collected to 1.8 Å resolution. Determination of the structure of HCHL will provide insight into the catalytic mechanism of an unusual enzymatic reaction with relevance to the applications of the enzyme in metabolic engineering.