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.
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[Show abstract][Hide abstract] ABSTRACT: The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.
Full-text · Article · Jan 2007 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: More than one activity: Owing to their hydratase activity, phenolic acid decarboxylases catalyze the regio- and stereoselective addition of H(2) O across the CC double bond of hydroxystyrene derivatives yielding (S)-4-(1-hydroxyethyl)phenols with up to 82 % conversion and 71 % ee. Based on structure analysis and molecular docking simulations a catalytic mechanism for this novel enzymatic reaction is proposed.
No preview · Article · Feb 2013 · Angewandte Chemie International Edition