A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism.
HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.
Bennett
Joseph P
JP
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, UK.
Bertin
Lucille
L
Moulton
Benjamin
B
Fairlamb
Ian J S
IJ
Brzozowski
A Marek
AM
Walton
Nicholas J
NJ
Grogan
Gideon
G
D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D001547:Benzaldehydes / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001547:Benzaldehydes; C119167:feruloyl-CoA; D000105:Acetyl Coenzyme A; C100058:vanillin; D005634:Fructose-Bisphosphate Aldolase; C119166:4-hydroxycinnamoyl-CoA hydratase-lyase; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase