title: Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism.
abstract: 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 P2(1)2(1)2 and unit-cell parameters a = 154.2, b = 167.5, c = 130.8 A. 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 A 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.
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, England.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Leonard
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Marshall
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Dodson
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Walton
authors.forename: Gideon
authors.initials: G
authors.lastname: Grogan
mesh_terms: D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry; D003373:Coumaric Acids / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004058:Diffusion; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification*; D008956:Models, Chemical; D015233:Models, Statistical; D011551:Pseudomonas fluorescens / Q000201:enzymology
chemical_list: D003373:Coumaric Acids; D002244:Carbon; C004999:ferulic acid; C119166:4-hydroxycinnamoyl-CoA hydratase-lyase; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase; D003065:Coenzyme A