D-alanine-(R)-lactate ligase

 

D-alanine--D-alanine ligase (EC:6.3.2.4) is a bacterial enzyme involved in cell-wall biosynthesis. The product of this enzyme, the depsipeptide D-alanyl-(R)-lactate, can be incorporated into the peptidoglycan pentapeptide instead of the usual D-alanyl-D-alanine dipeptide. The resulting peptidoglycan does not bind the glycopeptide antibiotics vancomycin and teicoplanin, conferring resistance on the bacteria.

 

Reference Protein and Structure

Sequence
P25051 UniProt (6.1.2.1) IPR005905 (Sequence Homologues) (PDB Homologues)
Biological species
Enterococcus faecium (Bacteria) Uniprot
PDB
1e4e - D-alanyl-D-lacate ligase (2.5 Å) PDBe PDBsum 1e4e
Catalytic CATH Domains
3.40.50.20 CATHdb 3.30.470.20 CATHdb (see all for 1e4e)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.1.2.1)

D-alanine zwitterion
CHEBI:57416ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
(R)-lactate
CHEBI:16004ChEBI
→
D-alanyl-(R)-lactic acid zwitterion
CHEBI:61166ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
Alternative enzyme names: VanA, VanB, VanD,

Enzyme Mechanism

Introduction

The D-lactate substrate attacks the terminal phosphate of ATP in a concerted mechanism to form ADP and phosphorylated D-lactate. D-alanine attacks the alpha phosphate of the D-lactate phosphate adduct, forming a tetrahedral intermediate. The tetrahedral intermediate collapses, eliminating inorganic phosphate and forming the D-alanyl-D-lactate adduct.

Catalytic Residues Roles

UniProt PDB* (1e4e)
His244 His244A His224 is responsible for pushing the equilibrium between D-lactate and D-alanyl binding in favour of the anionic D-lactate, with the positively charged imidazole group of His224 repelling the cationic amine groups of D-alanyl [PMID:10908650, PMID:18320587]. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
Gly311 (main-N), Arg290, His99 Gly311A (main-N), Arg290A, His99A Act to stabilise the negatively charged intermediates and transition states formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Tyr315, Glu250, Lys22 Tyr315A, Glu250A, Lys22A Forms a hydrogen bonding network that ensures His244 is in the correct protonation state. increase basicity, hydrogen bond donor
Val19 Val19A Ensures the correct stereoisomers are utilised. electrostatic stabiliser, steric role
*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

bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, intermediate collapse, native state of enzyme regenerated

References

  1. Wu D et al. (2008), Proteins, 72, 1148-1160. Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori. DOI:10.1002/prot.22009. PMID:18320587.
  2. Roper DI et al. (2000), Proc Natl Acad Sci U S A, 97, 8921-8925. The molecular basis of vancomycin resistance in clinically relevant Enterococci: Crystal structure of D-alanyl-D-lactate ligase (VanA). DOI:10.1073/pnas.150116497. PMID:10908650.
  3. Marshall CG et al. (1998), J Bacteriol, 180, 5792-5795. DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity. PMID:9791137.

Step 1. The D-lactate substrate attacks the terminal phosphate of ATP in a concerted mechanism to form ADP and phosphorylated D-lactate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly311A (main-N) hydrogen bond donor, electrostatic stabiliser
Lys22A hydrogen bond donor, increase basicity
His244A hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Val19A electrostatic stabiliser
Glu250A hydrogen bond acceptor, increase basicity
Arg290A attractive charge-charge interaction, electrostatic stabiliser
His99A attractive charge-charge interaction, electrostatic stabiliser
Tyr315A hydrogen bond donor, increase basicity

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed

Step 2. D-alanine attacks the alpha phosphate of the D-lactate phosphate adduct, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly311A (main-N) hydrogen bond donor, electrostatic stabiliser
Lys22A hydrogen bond donor
Val19A steric role, electrostatic stabiliser
Arg290A attractive charge-charge interaction, electrostatic stabiliser
His244A hydrogen bond donor, attractive charge-charge interaction, electrostatic stabiliser
Glu250A hydrogen bond acceptor
His99A hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Tyr315A hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 3. The tetrahedral intermediate collapses, eliminating inorganic phosphate and forming the D-alanyl-D-lactate adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly311A (main-N) hydrogen bond donor
Lys22A hydrogen bond donor
Val19A steric role
Arg290A attractive charge-charge interaction
His244A hydrogen bond donor
Glu250A hydrogen bond acceptor
His99A hydrogen bond donor
Tyr315A hydrogen bond donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The D-lactate substrate attacks the terminal phosphate of ATP in a concerted mechanism to form ADP and phosphorylated D-lactate.

Step 2. D-alanine attacks the alpha phosphate of the D-lactate phosphate adduct, forming a tetrahedral intermediate.

Step 3. The tetrahedral intermediate collapses, eliminating inorganic phosphate and forming the D-alanyl-D-lactate adduct.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday