The 1.8 A resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) from Pseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin.
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.
Leonard
Philip M
PM
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, England.
Brzozowski
A Marek
AM
Lebedev
Andrey
A
Marshall
Caroline M
CM
Smith
Derek J
DJ
Verma
Chandra S
CS
Walton
Nicholas J
NJ
Grogan
Gideon
G
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D001547:Benzaldehydes / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004746:Enoyl-CoA Hydratase / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D000214:Acyl Coenzyme A; D001547:Benzaldehydes; C119167:feruloyl-CoA; C022903:cinnamoyl-coenzyme A; C100058:vanillin; C119166:4-hydroxycinnamoyl-CoA hydratase-lyase; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase