Feruloyl CoA hydratase/lyase

 

Catalyses the hydration of the acyl-CoA thioester of ferulic acid and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde) and acetyl-CoA. This is a member of the crotonase superfamily.

 

Reference Protein and Structure

Sequence
O69762 UniProt (4.1.2.61) IPR001753 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas fluorescens (Bacteria) Uniprot
PDB
2vss - Wild-type Hydroxycinnamoyl-CoA hydratase lyase in complex with acetyl- CoA and vanillin (2.22 Å) PDBe PDBsum 2vss
Catalytic CATH Domains
3.90.226.10 CATHdb (see all for 2vss)
Click To Show Structure

Enzyme Reaction (EC:4.-.-.-)

water
CHEBI:15377ChEBI
+
feruloyl-CoA(4-)
CHEBI:57276ChEBI
→
acetyl-CoA(4-)
CHEBI:57288ChEBI
+
vanillin
CHEBI:18346ChEBI

Enzyme Mechanism

Introduction

In the first half-reaction (the hydrolysis of acyl-CoA thioester of ferulic acid) tautomerisation leads to the formation of a quinone-methide-enolate. This is followed by addition of a water molecule leading to the formation of the product of this half-reaction: 4-hydroxy-3-methoxyphenyl-Beta-hydroxypropiony-SCoA. The second half-reaction is the retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde) and acetyl-CoA.

Catalytic Residues Roles

UniProt PDB* (2vss)
Met70 (main-N), Gly120 (main-N) Met70A (main-N), Gly120A (main-N) Forms the oxyanion hole that binds the carbonyl group adjacent to the CoA sulfur. Act to stabilise the reactive intermediates and transition states formed at this position. hydrogen bond donor, electrostatic stabiliser
Tyr239, Tyr75 Tyr239B, Tyr75A Ionisation of the phenol group of feruloyl-CoA is induced by hydrogen bonding with the side chains of Tyr75 and Tyr239'. hydrogen bond donor, electrostatic stabiliser, steric role
Glu143 Glu143A Acts as a general acid/base, activating the nucleophilic water molecule for addition to the substrate. May also be involved in the final retro-aldol reaction that generates the acetyl-CoA and vanillin products. activator, proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

keto-enol tautomerisation, overall reactant used, intermediate formation, proton transfer, bimolecular nucleophilic addition, assisted keto-enol tautomerisation, unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, overall product formed, native state of enzyme regenerated

References

  1. Bennett JP et al. (2008), Biochem J, 414, 281-289. A ternary complex of hydroxycinnamoyl-CoA hydratase–lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism. DOI:10.1042/bj20080714. PMID:18479250.
  2. Ma G et al. (2014), Int J Quantum Chem, 114, 249-254. A density functional theory study on the catalytic mechanism of hydroxycinnamoyl-CoA hydratase-lyase. DOI:10.1002/qua.24551.
  3. Botosoa EP et al. (2009), Anal Biochem, 393, 182-188. Quantitative isotopic 13C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: the case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin. DOI:10.1016/j.ab.2009.06.031. PMID:19563771.
  4. Leonard PM et al. (2006), Acta Crystallogr D Biol Crystallogr, 62, 1494-1501. 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. DOI:10.1107/S0907444906039199. PMID:17139085.
  5. Gasson MJ et al. (1998), J Biol Chem, 273, 4163-4170. Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester. PMID:9461612.

Step 1. Ionisation of the phenol group of feruloyl-CoA is induced by hydrogen bonding with the side chains of Tyr75 and Tyr239'. A tautomerisation leads to the formation of a quinone-methide-enolate which is stabilised in the oxyanion hole formed by the backbone amides of Met70 and Gly120

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Catalytic Residues Roles

Residue Roles
Met70A (main-N) electrostatic stabiliser, hydrogen bond donor
Gly120A (main-N) electrostatic stabiliser, hydrogen bond donor
Tyr75A electrostatic stabiliser, hydrogen bond donor, steric role
Tyr239B electrostatic stabiliser, hydrogen bond donor, steric role

Chemical Components

keto-enol tautomerisation, overall reactant used, intermediate formation

Step 2. The quinone-methide-enolate is the substrate for hydration by a water molecule activated by Glu143.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met70A (main-N) electrostatic stabiliser, hydrogen bond donor
Gly120A (main-N) electrostatic stabiliser, hydrogen bond donor
Tyr75A electrostatic stabiliser, hydrogen bond donor, steric role
Tyr239B electrostatic stabiliser, hydrogen bond donor, steric role
Glu143A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 3. Glu-143 acts as acid to protonate the double bond leading to the intermediate 4-hydroxy-3-methoxyphenyl-Beta-hydroxypropiony-SCoA

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met70A (main-N) electrostatic stabiliser, hydrogen bond donor
Gly120A (main-N) electrostatic stabiliser, hydrogen bond donor
Tyr75A electrostatic stabiliser, hydrogen bond donor
Tyr239B electrostatic stabiliser, hydrogen bond donor
Glu143A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation

Step 4. A retro-aldol reaction generates acetyl-CoA and vanillin. Glu143 may act as a catalytic base. The reaction might also be favoured by strain effects exerted by the binding of the phenolic hydroxyl by Tyr75 and Tyr239'.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met70A (main-N) electrostatic stabiliser, hydrogen bond donor
Gly120A (main-N) electrostatic stabiliser, hydrogen bond donor
Tyr75A electrostatic stabiliser, hydrogen bond donor
Tyr239B electrostatic stabiliser, hydrogen bond donor
Glu143A activator

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Ionisation of the phenol group of feruloyl-CoA is induced by hydrogen bonding with the side chains of Tyr75 and Tyr239'. A tautomerisation leads to the formation of a quinone-methide-enolate which is stabilised in the oxyanion hole formed by the backbone amides of Met70 and Gly120

Step 2. The quinone-methide-enolate is the substrate for hydration by a water molecule activated by Glu143.

Step 3. Glu-143 acts as acid to protonate the double bond leading to the intermediate 4-hydroxy-3-methoxyphenyl-Beta-hydroxypropiony-SCoA

Step 4. A retro-aldol reaction generates acetyl-CoA and vanillin. Glu143 may act as a catalytic base. The reaction might also be favoured by strain effects exerted by the binding of the phenolic hydroxyl by Tyr75 and Tyr239'.

Products.

Contributors

Daniel E. Almonacid, Sophie T. Williams, Gemma L. Holliday, James Willey