L-Ala-D/L-Glu epimerase

 

L-Ala-D/L-Glu epimerase, first characterised from the bacteria Escherichia coli and Bacillus subtilis, catalyses the epimerisation of L-Ala-D-Glu to L-Ala-L-Glu and is involved in the recycling of the murein peptide, of which L-Ala-D-Glu is a component. In vitro the enzyme from Escherichia coli has been shown to epimerise several L-Ala-L-X dipeptides with broader specificity than the enzyme from Bacillus subtilis.

 

Reference Protein and Structure

Sequence
O34508 UniProt (5.1.1.20) IPR034390 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1tkk - The Structure of a Substrate-Liganded Complex of the L-Ala-D/L-Glu Epimerase from Bacillus subtilis (2.1 Å) PDBe PDBsum 1tkk
Catalytic CATH Domains
3.20.20.120 CATHdb (see all for 1tkk)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:5.1.1.20)

L-alanyl-L-glutamate(1-)
CHEBI:61396ChEBI
→
L-alanyl-D-glutamate(1-)
CHEBI:61395ChEBI
Alternative enzyme names: YkfB, YcjG, AEE, AE epimerase,

Enzyme Mechanism

Introduction

As a member of the Enolase Superfamily, this enzyme is thought to proceed with the characteristic abstraction of the proton alpha to the carboxylate group of the substrate that is stabilised by coordination to the essential Mg(II) ion. It has been shown that the reaction is accompanied by incorporation of a solvent-derived hydrogen into the product. In this reversible reaction, one of the conserved lysine residues abstracts the alpha proton and the other lysine protonates the enolate anion intermediate.

Catalytic Residues Roles

UniProt PDB* (1tkk)
Lys162 Lys162A Acts as a general acid/base. Abstracts the proton alpha to the carboxyl group during the (R)-substrate epimerisation. proton acceptor, proton donor
Asp191, Glu219, Asp244 Asp191A, Glu219A, Asp244A Forms part of the magnesium binding site. metal ligand
Lys268 Lys268A Acts as a general acid/base. Abstracts the proton alpha to the carboxyl group during the (S)-substrate epimerisation. 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, proton transfer, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Schmidt DMZ et al. (2001), Biochemistry, 40, 15707-15715. Evolution of Enzymatic Activities in the Enolase Superfamily:  Functional Assignment of Unknown Proteins inBacillus subtilisandEscherichia coliasl-Ala-d/l-Glu Epimerases†. DOI:10.1021/bi011640x.
  2. Klenchin VA et al. (2004), Biochemistry, 43, 10370-10378. Evolution of enzymatic activities in the enolase superfamily: structure of a substrate-liganded complex of the L-Ala-D/L-Glu epimerase from Bacillus subtilis. DOI:10.1021/bi049197o. PMID:15301535.
  3. Gulick AM et al. (2001), Biochemistry, 40, 15716-15724. Evolution of enzymatic activities in the enolase superfamily: crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis. PMID:11747448.

Step 1. Lys268 abstracts the proton alpha to the carboxylate group, forming a planar intermediate which is stabilised by the magnesium ion. In the reverse reaction, Lys162 abstracts the proton from the substrate.

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Catalytic Residues Roles

Residue Roles
Asp191A metal ligand
Glu219A metal ligand
Asp244A metal ligand
Lys268A proton acceptor

Chemical Components

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

Step 2. The planar intermediate collapses. Lys162 donates a proton to the intermediate to form the (R)-stereoisomer. In the reverse reaction, Lys268 performs this function.

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Catalytic Residues Roles

Residue Roles
Asp191A metal ligand
Glu219A metal ligand
Asp244A metal ligand
Lys162A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, overall product formed

Step 3. The incorporation of duterium from D2O suggests that water is the source of the proton required to return the enzyme to its original starting protonation state. However, this step is optional as the enzyme catalyses the epimerisation reaction in both directions.

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Catalytic Residues Roles

Residue Roles
Asp191A metal ligand
Glu219A metal ligand
Asp244A metal ligand
Lys268A proton donor
Lys162A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Lys268 abstracts the proton alpha to the carboxylate group, forming a planar intermediate which is stabilised by the magnesium ion. In the reverse reaction, Lys162 abstracts the proton from the substrate.

Step 2. The planar intermediate collapses. Lys162 donates a proton to the intermediate to form the (R)-stereoisomer. In the reverse reaction, Lys268 performs this function.

Step 3. The incorporation of duterium from D2O suggests that water is the source of the proton required to return the enzyme to its original starting protonation state. However, this step is optional as the enzyme catalyses the epimerisation reaction in both directions.

Products.

Contributors

Gemma L. Holliday