Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism.
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.
Leonard
Philip M
PM
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, England.
Marshall
Caroline M
CM
Dodson
Eleanor J
EJ
Walton
Nicholas J
NJ
Grogan
Gideon
G
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
D003373:Coumaric Acids; D002244:Carbon; C004999:ferulic acid; C119166:4-hydroxycinnamoyl-CoA hydratase-lyase; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase; D003065:Coenzyme A