2-dehydro-3-deoxyglucarate aldolase

 

5-keto-4-deoxy-D-glucarate aldolase GarL is involved in step 2 of the subpathway that synthesizes D-glycerate from galactarate. It catalyses the reversible retro-aldol cleavage of both 5-keto-4-deoxy-D-glucarate and 2-keto-3-deoxy-D-glucarate to pyruvate and tartronic semialdehyde.

 

Reference Protein and Structure

Sequence
P23522 UniProt (4.1.2.20) IPR017648 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1dxe - 2-dehydro-3-deoxy-galactarate aldolase from Escherichia coli (1.8 Å) PDBe PDBsum 1dxe
Catalytic CATH Domains
3.20.20.60 CATHdb (see all for 1dxe)
Cofactors
Magnesium(2+) (1), Water (1)
Click To Show Structure

Enzyme Reaction (EC:4.1.2.20)

2-dehydro-3-deoxy-D-glucarate(2-)
CHEBI:58098ChEBI
→
pyruvate
CHEBI:15361ChEBI
+
2-hydroxy-3-oxopropanoate
CHEBI:57978ChEBI
Alternative enzyme names: 2-keto-3-deoxyglucarate aldolase, Alpha-keto-beta-deoxy-D-glucarate aldolase, 2-dehydro-3-deoxy-D-glucarate tartronate-semialdehyde-lyase,

Enzyme Mechanism

Introduction

The Asp89-His50 intersubunit dyad deprotonates the metal-bound water molecule. The condensed substrate binds to the metal ion in a bidentate fashion with the C3 carbonyl and C4 hydroxyl interacting with Arg75. The metal-bound hydroxide ion, activated by the intersubunit Asp89-His50 dyad, abstracts the C4 hydroxyl proton, coupled with breakage of the C3−C4 carbon−carbon bond. The resulting aldehyde product and pyruvate enolate are stabilised by the metal ion and Arg74. Protonation of pyruvate enolate at C3 by the Asp89-His50-metal-bound water results in the other product pyruvate, returning the enzyme to the ground state and completing the catalytic cycle.

Catalytic Residues Roles

UniProt PDB* (1dxe)
Arg75 Arg75A Helps stabilise the transition state. electrostatic stabiliser
Glu153, Asp179 Glu153A, Asp179A Forms part of the magnesium binding site. metal ligand
His50 His50A Acts as a general acid/base. proton acceptor, proton donor
Asp89 Asp89A(AA) Increases basicity of active site His50. modifies pKa, 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, bimolecular elimination, overall product formed, overall reactant used, assisted keto-enol tautomerisation, native state of enzyme regenerated

References

  1. Rea D et al. (2008), Biochemistry, 47, 9955-9965. Crystal structure and functional assignment of YfaU, a metal ion dependent class II aldolase from Escherichia coli K12. DOI:10.1021/bi800943g. PMID:18754683.
  2. Wang W et al. (2008), FEBS Lett, 582, 3385-3388. The role of a conserved histidine residue in a pyruvate-specific Class II aldolase. DOI:10.1016/j.febslet.2008.08.032. PMID:18775708.
  3. Rea D et al. (2007), J Mol Biol, 373, 866-876. Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli. DOI:10.1016/j.jmb.2007.06.048. PMID:17881002.
  4. Wang W et al. (2005), Biochemistry, 44, 9447-9455. Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI. DOI:10.1021/bi050607y. PMID:15996099.
  5. Izard T et al. (2000), EMBO J, 19, 3849-3856. Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism. DOI:10.1093/emboj/19.15.3849. PMID:10921867.
  6. Hubbard BK et al. (1998), Biochemistry, 37, 14369-14375. Evolution of Enzymatic Activities in the Enolase Superfamily:  Characterization of the (D)-Glucarate/Galactarate Catabolic Pathway inEscherichia coli†. DOI:10.1021/bi981124f. PMID:9772162.

Step 1. His50 abstracts a proton from a metal-bound water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp89A(AA) modifies pKa
Arg75A electrostatic stabiliser
Asp179A metal ligand
Glu153A metal ligand
His50A proton acceptor

Chemical Components

proton transfer

Step 2. The activated water abstracts a proton from the substrate, forming the aldehyde product and the pyruvoyl intermediate.

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Catalytic Residues Roles

Residue Roles
Arg75A electrostatic stabiliser
Asp89A(AA) electrostatic stabiliser
Asp179A metal ligand
Glu153A metal ligand

Chemical Components

proton transfer, ingold: bimolecular elimination, overall product formed, overall reactant used

Step 3. The enolate intermediate collapses with concomitant deprtonation of the water molecule, which in turn abstracts a proton from His50.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp89A(AA) modifies pKa
Arg75A electrostatic stabiliser
Asp179A metal ligand
Glu153A metal ligand
His50A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. His50 abstracts a proton from a metal-bound water.

Step 2. The activated water abstracts a proton from the substrate, forming the aldehyde product and the pyruvoyl intermediate.

Step 3. The enolate intermediate collapses with concomitant deprtonation of the water molecule, which in turn abstracts a proton from His50.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday