D-Ala-D-Ala dipeptidase

 

VanX is a Zn(II)-containing metalloenzyme that cleaves D-alanyl-D-alanine dipeptide (D-ala-D-ala), increasing the cytoplasmic pool of free D-ala available for use by VanA to form D-ala-D-lac. The enzyme is stereospecific and is found in solution as a mixture of monomers and dimers. The dipeptidase is a critical component of a system that mediates transposon-based, high-level vancomycin resistance in enterococci. Vancomycin acts by binding to the terminal D-ala-D-ala moiety of the bacterial peptidoglycan precursor, interfering with growth of the peptidoglycan chain and normal cell wall formation. The substitution of D-ala-D-ala by D-ala-D-lac at the extracellular terminus of the peptidoglycan chain diminishes the affinity of vancomycin by 1000-fold.

 

Reference Protein and Structure

Sequence
Q06241 UniProt (3.4.13.22) IPR000755 (Sequence Homologues) (PDB Homologues)
Biological species
Enterococcus faecium (Bacteria) Uniprot
PDB
1r44 - Crystal Structure of VanX (2.25 Å) PDBe PDBsum 1r44
Catalytic CATH Domains
3.30.1380.10 CATHdb (see all for 1r44)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.4.13.22)

water
CHEBI:15377ChEBI
+
D-alanyl-D-alanine zwitterion
CHEBI:57822ChEBI
→
D-alanine zwitterion
CHEBI:57416ChEBI
Alternative enzyme names: D-alanyl-D-alanine dipeptidase, VanX D-Ala-D-Ala dipeptidase, VanX,

Enzyme Mechanism

Introduction

An incoming peptide displaces a zinc-bound water molecule towards Glu181, giving a six-coordinated metal ion. The nucleophilic hydroxide is generated by zinc and Glu181 from the water molecule, and the nucleophile attacks the polarised carbonyl to form a tetrahedral intermediate. This forms a bidentate complex with the zinc and is stabilised further by interactions with Arg71. It is likely that Glu181 donates a proton to the scissile nitrogen, completing the reaction and yielding D-ala, although it is possible that the source of the proton may be from the nucleophile or another active site residue.

Catalytic Residues Roles

UniProt PDB* (1r44)
His116, Asp123, His184 His116A, Asp123A, His184A Form the Zinc binding site. metal ligand
Arg71 Arg71A Arg71 is a key residue in stabilising the tetrahedral transition state by formation of a salt link to one of the phosphonate oxygens. electrostatic stabiliser
Glu181 Glu181A Glu181 acts as a catalytic base, deprotonating the water molecule to form the nucleophile. The residue is thought to donate a proton to the scissile nitrogen to complete the reaction. 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

proton transfer, bimolecular nucleophilic addition, coordination to a metal ion, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, decoordination from a metal ion, native state of enzyme regenerated, overall product formed

References

  1. Matthews ML et al. (2006), J Am Chem Soc, 128, 13050-13051. Probing the Reaction Mechanism of thed-ala-d-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme. DOI:10.1021/ja0627343. PMID:17017774.
  2. Meziane-Cherif D et al. (2014), Proc Natl Acad Sci U S A, 111, 5872-5877. Structural basis for the evolution of vancomycin resistance D,D-peptidases. DOI:10.1073/pnas.1402259111. PMID:24711382.
  3. Breece RM et al. (2005), J Biol Chem, 280, 11074-11081. A five-coordinate metal center in Co(II)-substituted VanX. DOI:10.1074/jbc.M412582200. PMID:15657055.
  4. Bussiere DE et al. (1998), Mol Cell, 2, 75-84. The Structure of VanX Reveals a Novel Amino-Dipeptidase Involved in Mediating Transposon-Based Vancomycin Resistance. DOI:10.1016/s1097-2765(00)80115-x. PMID:9702193.
  5. McCafferty DG et al. (1997), Biochemistry, 36, 10498-10505. Mutational Analysis of Potential Zinc-Binding Residues in the Active Site of the Enterococcald-Ala-d-Ala Dipeptidase VanX†. DOI:10.1021/bi970543u. PMID:9265630.

Step 1. Glu181 deprotonates a water which alongside Zn polarising the hydroxide activates it so it can attack the carbon of the carbonyl in a nucleophilic addition. The incoming pe-ptide also coordinates to the Zinc which further stabilises the tetrahedral intermediate produced.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg71A electrostatic stabiliser
His116A metal ligand
Asp123A metal ligand
His184A metal ligand
Glu181A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, coordination to a metal ion, intermediate formation, overall reactant used, rate-determining step

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from Glu181.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His116A metal ligand
Asp123A metal ligand
His184A metal ligand
Arg71A electrostatic stabiliser
Glu181A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, decoordination from a metal ion, native state of enzyme regenerated

Step 3. The N-terminal product accepts a proton from the C-terminal product to produce the kinetically favourable products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His116A metal ligand
Asp123A metal ligand
His184A metal ligand
Arg71A electrostatic stabiliser

Chemical Components

proton transfer, overall product formed
Download: Image, Marvin File

Step 1. Glu181 deprotonates a water which alongside Zn polarising the hydroxide activates it so it can attack the carbon of the carbonyl in a nucleophilic addition. The incoming pe-ptide also coordinates to the Zinc which further stabilises the tetrahedral intermediate produced.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the peptide bond. The N-terminal product then accepts a proton from Glu181.

Step 3. The N-terminal product accepts a proton from the C-terminal product to produce the kinetically favourable products.

Products.

Contributors

Gemma L. Holliday, Charity Hornby