2VSU: A Ternary Complex Of Hydroxycinnamoyl-Coa Hydratase-Lyase ( Hchl) With Acetyl-Coenzyme A And Vanillin Gives Insights Into Substrate Specificity And Mechanism

Citation:
Abstract
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.
PDB ID: 2VSUDownload
MMDB ID: 64654
PDB Deposition Date: 2008/4/29
Updated in MMDB: 2011/08
Experimental Method:
x-ray diffraction
Resolution: 1.9  Å
Source Organism:
Similar Structures:
Biological Unit for 2VSU: hexameric; determined by author and by software (PISA)
Molecular Components in 2VSU
Label Count Molecule
Proteins (6 molecules)
3
P-hydroxycinnamoyl COA Hydratase/lyase
Molecule annotation
1
P-hydroxycinnamoyl COA Hydratase/lyase
Molecule annotation
1
P-hydroxycinnamoyl COA Hydratase/lyase
Molecule annotation
1
P-hydroxycinnamoyl COA Hydratase/lyase
Molecule annotation
Chemicals (6 molecules)
1
5
2
1
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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