D(-)-tartrate dehydratase
D-tartrate dehydratase (TarD), isolated from Bradyrhizobium japonicum, catalyses the dehydration of D-tartrate to oxaloacetate and water. TarD is a member of the mandelate racemase subgroup of the enolase superfamily.
Reference Protein and Structure
- Sequence
-
Q89FH0
(4.2.1.81)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Bradyrhizobium diazoefficiens USDA 110 (Bacteria)
- PDB
-
2dw7
- Crystal structure of D-tartrate dehydratase from Bradyrhizobium japonicum complexed with Mg++ and meso-tartrate
(2.5 Å)
- Catalytic CATH Domains
-
3.30.390.10
3.20.20.120
(see all for 2dw7)
- Cofactors
- Magnesium(2+) (1)
Enzyme Reaction (EC:4.2.1.81)
→
+
Alternative enzyme names: D-tartrate dehydratase, (S,S)-tartrate hydro-lyase,
Enzyme Mechanism
Introduction
Reaction mechanism is a simple extension of the two-step reaction catalysed by other members of the enolase superfamily: Lys-184 initiates the reaction by abstraction of the alpha-proton to generate a Mg(II)-stabilised enediolate intermediate, and the vinylogous beta-elimination of the 3-OH group is general acid-catalysed by the His-322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereo-random, suggesting that the enol tautomer of oxaloacetate is the product.
Catalytic Residues Roles
| UniProt | PDB* (2dw7) | ||
| Asp213, Glu265, Glu239 | Asp213A, Glu265A, Glu239A | Forms the magnesium binding site. | metal ligand |
| Asn55 | Asn55A | Forms a hydrogen bond to the 3-hydroxyl leaving group. This is likely to stabilise the transition state of vinylogous beta-elimination. | electrostatic stabiliser |
| Lys182 | Lys182A | Interacts with one of the carboxylate oxygens of the substrate. Increased electrostatic interactions with the carboxylate during enolisation stabilises the transition state. | electrostatic stabiliser |
| Lys184 | Lys184A | Abstracts the alpha-proton from D-tartarate, initiating enolisation. | proton acceptor, proton donor |
| Asp292 | Asp292A | Forms a hydrogen bond to the general acid/base histidine to form a catalytic dyad. This is thought modulate the pKa of the catalytic histidine. | increase basicity, modifies pKa, electrostatic stabiliser |
| His322 | His322A | Protonates the 3-hydroxyl leaving group during vinylogous beta-elimination. | proton acceptor, proton donor |
| Glu341 | Glu341A | Forms a hydrogen bond to one of the carboxylate oxygens of the substrate. The strengthening of this hydrogen bond upon enolisation stabilises the transition state. | electrostatic stabiliser |
*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, unimolecular elimination by the conjugate base, dehydration, inferred reaction stepReferences
- Yew WS et al. (2006), Biochemistry, 45, 14598-14608. Evolution of Enzymatic Activities in the Enolase Superfamily: d-Tartrate Dehydratase fromBradyrhizobium japonicum†,‡. DOI:10.1021/bi061688g. PMID:17144653.
- Prat-Resina X et al. (2005), J Phys Chem B, 109, 21089-21101. Reaction Mechanism of the Mandelate Anion Racemization Catalyzed by Mandelate Racemase Enzyme: A QM/MM Molecular Dynamics Free Energy Study. DOI:10.1021/jp052239d. PMID:16853732.
- Wieczorek SJ et al. (1999), J Am Chem Soc, 121, 4540-4541. Evolution of Enzymatic Activities in the Enolase Superfamily: Identification of a “New” General Acid Catalyst in the Active Site ofd-Galactonate Dehydratase fromEscherichia coli. DOI:10.1021/ja990500w.
- Schafer SL et al. (1996), Biochemistry, 35, 5662-5669. Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the D270N Mutant†,‡. DOI:10.1021/bi960174m. PMID:8639525.
- Kallarakal AT et al. (1995), Biochemistry, 34, 2788-2797. Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the K166R Mutant. DOI:10.1021/bi00009a007. PMID:7893690.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Asn55A | electrostatic stabiliser |
| Lys182A | electrostatic stabiliser |
| Asp292A | electrostatic stabiliser, modifies pKa |
| Glu341A | electrostatic stabiliser |
| Glu265A | metal ligand |
| Glu239A | metal ligand |
| Asp213A | metal ligand |
| Lys184A | proton acceptor |
Chemical Components
proton transfer, assisted keto-enol tautomerisation
Step 2. The intermediate collapses, eliminating water which is formed by abstraction of a proton from His322.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu265A | metal ligand |
| Glu239A | metal ligand |
| Asp213A | metal ligand |
| Asn55A | electrostatic stabiliser |
| Lys182A | electrostatic stabiliser |
| Glu341A | electrostatic stabiliser |
| Asp292A | electrostatic stabiliser |
| His322A | proton donor |
Chemical Components
proton transfer, ingold: unimolecular elimination by the conjugate base, dehydration
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu265A | metal ligand |
| Glu239A | metal ligand |
| Asp213A | metal ligand |
| Asp292A | increase basicity |
| His322A | proton acceptor |
| Lys184A | proton donor |
Chemical Components
proton transfer, inferred reaction step
Catalytic Residues Roles
| Residue | Roles |
|---|
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