Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism

TY - JOUR

T1 - Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism

AU - Leonard, P M

AU - Marshall, C M

AU - Dodson, E J

AU - Walton, N J

AU - Grogan, G

PY - 2004/12

Y1 - 2004/12

N2 - 4-Hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), also called feruloyl-CoA hydratase- lyase (FCHL), from Pseudomonas uorescens 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 Angstrom. 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 Angstrom 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.

AB - 4-Hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), also called feruloyl-CoA hydratase- lyase (FCHL), from Pseudomonas uorescens 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 Angstrom. 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 Angstrom 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.

KW - FERULIC ACID

KW - 4-HYDROXYCINNAMOYL-COA HYDRATASE/LYASE

KW - DELTA(3)-DELTA(2)-ENOYL-COA ISOMERASE

KW - ANGSTROM RESOLUTION

KW - CRYSTAL-STRUCTURE

KW - ENZYME

KW - SUPERFAMILY

KW - PSEUDOMONAS

KW - CLEAVAGE

KW - MODE

U2 - 10.1107/S0907444904024588

DO - 10.1107/S0907444904024588

M3 - Article

SN - 0907-4449

VL - 60

SP - 2343

EP - 2345

JO - Acta Crystallographica. Section D, Biological Crystallography

JF - Acta Crystallographica. Section D, Biological Crystallography

ER -