4-hydroxybutanoyl-CoA dehydratase

 

4-hydroxybutyryl-CoA dehydratase (4-BUDH) catalyses the dehydration of 4-hydroxybutanoyl-CoA to crotonyl-CoA, as well as the isomerisation of vinylacetyl-CoA to crotonyl-CoA. The reaction is mechanistically the most demanding step in the fermentation of gamma-aminobutyrate to ammonia, acetate and butyrate by Clostridium aminobutyricum.

 

Reference Protein and Structure

Sequence
P55792 UniProt (4.2.1.120, 5.3.3.3) IPR004925 (Sequence Homologues) (PDB Homologues)
Biological species
Clostridium aminobutyricum (Bacteria) Uniprot
PDB
1u8v - Crystal Structure of 4-Hydroxybutyryl-CoA Dehydratase from Clostridium aminobutyricum: Radical catalysis involving a [4Fe-4S] cluster and flavin (1.6 Å) PDBe PDBsum 1u8v
Catalytic CATH Domains
1.20.140.10 CATHdb 1.10.3140.10 CATHdb 2.40.110.10 CATHdb (see all for 1u8v)
Cofactors
Fadh2(2-) (1), Tetra-mu3-sulfido-tetrairon (1)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.120)

4-hydroxybutyryl-CoA(4-)
CHEBI:58574ChEBI
→
water
CHEBI:15377ChEBI
+
crotonoyl-CoA(4-)
CHEBI:57332ChEBI

Enzyme Mechanism

Introduction

4-BUDH utilises an oxidised FAD quinone to carry out radical formation. This lowers the pKa of the C3 hydrogen, thus activating the usually unreactive C3-H bond. His 292 acts as a general base to enolise 4-hydroxybutanoyl-CoA by abstraction of the C2-proR-hydrogen. FAD removes an electron from the enolate oxygen to create the enoxy radical. The FAD-. semiquinone radical anion abstracts the substrate's C3-proS-hydrogen. The substrate is now a ketyl radical anion with the hydroxy group coordinated to an iron in the Fe-S cluster. This polarises the hydroxy group and enables the ketyl radical anion to eliminate water, with the extra proton coming from the FADH. semiquinone (i.e. the proton that was removed from C3 by FAD-.). The dienoxy radical produced undergoes one electron reduction by FAD-. to yield a dienolate. Glu455 donates a proton to C4 of the dienolate to yield the product.

Catalytic Residues Roles

UniProt PDB* (1u8v)
Glu455 Glu455A Aids in the elimination of water at C4 and reprotonates C4 to generate the product. proton donor
Glu257, Thr190 Glu257A, Thr190A Deprotonates the neutral semiquinone to regenerate the semiquinone anion proton acceptor
Cys103, Cys299, Cys99 Cys103A, Cys299A, Cys99A Bond to the iron sulfur cluster
His292 His292A His 292 is one of the ligands to the Fe-S cluster. It acts as a general acid/base to the substrate, converting substrate to the enol (deprotonating C2), and the dienolate to the product (protonating C4). proton acceptor
*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

proton transfer, assisted keto-enol tautomerisation, overall reactant used, radical formation, intramolecular elimination, radical termination, native state of cofactor regenerated, overall product formed

References

  1. Martins BM et al. (2004), Proc Natl Acad Sci U S A, 101, 15645-15649. Crystal structure of 4-hydroxybutyryl-CoA dehydratase: Radical catalysis involving a [4Fe-4S] cluster and flavin. DOI:10.1073/pnas.0403952101. PMID:15496473.
  2. Buckel W et al. (2012), Biochim Biophys Acta, 1824, 1278-1290. Enzyme catalyzed radical dehydrations of hydroxy acids. DOI:10.1016/j.bbapap.2011.11.009. PMID:22178228.

Step 1. His292 abstracts a proton from C2 this causes enolisation.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
His292A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall reactant used

Step 2. FAD abstracts an electron from the enolate oxygen forming enoxy and semiquinone radicals.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
Tyr296A radical stabiliser

Chemical Components

radical formation

Step 3. The semiquinone anion abstracts a proton from C3, leading to the formation of a ketyl radical anion and a neutral semiquinone.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
Tyr296A radical stabiliser

Chemical Components

proton transfer

Step 4. The ketyl radical ion eliminates water at the C4 position to form a dienoxy radical, this is aided by protonation from Glu455. Thr190 and Glu257 both act to deprotonate the neutral semiquinone, reforming the semiquinone anion.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
Tyr296A radical stabiliser
Thr190A proton relay
Glu257A proton acceptor
Glu455A proton donor
Thr190A proton acceptor, proton donor

Chemical Components

ingold: intramolecular elimination, proton transfer

Step 5. The dienoxy radical abstracts an electron from the semiquinone forming a dienolate and quinone.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
Tyr296A radical stabiliser

Chemical Components

radical termination, native state of cofactor regenerated

Step 6. The dienolate tautomerizes via protonation at C4 from Glu455 to form the product crotonyl-CoA. It is not clear how Glu455 is protonated between this and the previous step.

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Catalytic Residues Roles

Residue Roles
Tyr296A electrostatic stabiliser
Glu455A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, overall product formed
Download: Image, Marvin File

Step 1. His292 abstracts a proton from C2 this causes enolisation.

Step 2. FAD abstracts an electron from the enolate oxygen forming enoxy and semiquinone radicals.

Step 3. The semiquinone anion abstracts a proton from C3, leading to the formation of a ketyl radical anion and a neutral semiquinone.

Step 4. The ketyl radical ion eliminates water at the C4 position to form a dienoxy radical, this is aided by protonation from Glu455. Thr190 and Glu257 both act to deprotonate the neutral semiquinone, reforming the semiquinone anion.

Step 5. The dienoxy radical abstracts an electron from the semiquinone forming a dienolate and quinone.

Step 6. The dienolate tautomerizes via protonation at C4 from Glu455 to form the product crotonyl-CoA. It is not clear how Glu455 is protonated between this and the previous step.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, James Willey