Galactarate dehydratase (D-threo-forming)

 

Galactarate dehydratase (D-threo-forming) enzymes catalyse the dehydration of galactarate. The product of this reaction is the enantiomer of the product of the galactarate dehydratase reaction catalysed by the L-talarate/galactarate dehydratase family in the mandelate racemase subgroup.

 

Reference Protein and Structure

Sequence
Q8EMJ9 UniProt (4.2.1.158) IPR033977 (Sequence Homologues) (PDB Homologues)
Biological species
Oceanobacillus iheyensis HTE831 (Bacteria) Uniprot
PDB
3es7 - Crystal structure of divergent enolase from Oceanobacillus Iheyensis complexed with Mg and L-malate. (1.9 Å) PDBe PDBsum 3es7
Catalytic CATH Domains
3.30.390.10 CATHdb 3.20.20.120 CATHdb (see all for 3es7)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.158)

galactarate(2-)
CHEBI:16537ChEBI
→
3-deoxy-D-threo-hex-2-ulosarate(2-)
CHEBI:78267ChEBI
+
water
CHEBI:15377ChEBI

Enzyme Mechanism

Introduction

A Tyr-Arg dyad is responsible for the abstraction of the proton alpha to the carbonyl group in the first step of a classical enolase reaction. A second tyrosine acts as the general acid in the second step, which results in the elimination of a water molecule. The final step of the reaction is an assisted keto-enol tautomerisation that is thought to be initiated by Tyr90, and regenerates the enzyme's ground state.

Catalytic Residues Roles

UniProt PDB* (3es7)
Arg162 Arg162A Part of the Arg-Tyr dyad, it s responsible for perturbing the pKa of Tyr164 such that it can act as the initial general base. modifies pKa, electrostatic stabiliser
Tyr164 Tyr164A Acts as a general acid/base. This tyrosine is activated as part of an Arg-Tyr dyad. It is responsible for the initial deprotonation if the galactarate C2 atom. proton acceptor, proton donor
His246, Asp193, Glu221 His246A, Asp193A, Glu221A Forms the magnesium 2 binding site. This is the classical enolase metal binding site. metal ligand
Thr297, Asp42, His45 Thr297A, Asp42A, His45A Form the magnesium 1 binding site. metal ligand
Tyr90 Tyr90A Acts as a general acid/base. This tyrosine is on the opposite side of the substrate to Tyr164 and is responsible for facilitating the departure of beta-hydroxide leaving group. It also acts as the acid catalyst for ketonization of enol intermediate. 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

assisted keto-enol tautomerisation, dehydration, unimolecular elimination by the conjugate base, native state of enzyme regenerated

References

  1. Rakus JF et al. (2009), Biochemistry, 48, 11546-11558. Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase fromOceanobacillus iheyensis,. DOI:10.1021/bi901731c. PMID:19883118.

Step 1. Tyr164, activated by Arg162, abstracts the C2 proton from the galactarate substrate.

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Catalytic Residues Roles

Residue Roles
Arg162A modifies pKa
Asp193A metal ligand
Glu221A metal ligand
His45A metal ligand
Asp42A metal ligand
Thr297A metal ligand
His246A metal ligand
Arg162A electrostatic stabiliser
Tyr164A proton acceptor

Chemical Components

assisted keto-enol tautomerisation

Step 2. The negatively charged intermediate collapses, eliminating water (with concomitant deprotonation of Tyr90).

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Catalytic Residues Roles

Residue Roles
Asp193A metal ligand
Glu221A metal ligand
His45A metal ligand
Asp42A metal ligand
Thr297A metal ligand
His246A metal ligand
Tyr90A proton donor

Chemical Components

assisted keto-enol tautomerisation, dehydration, ingold: unimolecular elimination by the conjugate base

Step 3. Tyr90 deprotonates the OH group of the interemdiate in an assisted keto-enol tautomerisation. This results in the regeneration of the active site and production of the final product.

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Catalytic Residues Roles

Residue Roles
Arg162A modifies pKa
Asp193A metal ligand
Glu221A metal ligand
His45A metal ligand
Asp42A metal ligand
Thr297A metal ligand
His246A metal ligand
Tyr164A proton donor
Tyr90A proton acceptor

Chemical Components

assisted keto-enol tautomerisation, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Tyr164, activated by Arg162, abstracts the C2 proton from the galactarate substrate.

Step 2. The negatively charged intermediate collapses, eliminating water (with concomitant deprotonation of Tyr90).

Step 3. Tyr90 deprotonates the OH group of the interemdiate in an assisted keto-enol tautomerisation. This results in the regeneration of the active site and production of the final product.

Products.

Contributors

Gemma L. Holliday, Shoshana Brown