D-alanine---D-alanine ligase

 

D-alanine--D-alanine ligase (EC:6.3.2.4) is a bacterial enzyme involved in cell-wall biosynthesis. It participates in forming UDP-N-acetylmuramoyl pentapeptide, the peptidoglycan precursor.

 

Reference Protein and Structure

Sequence
P07862 UniProt (6.3.2.4) IPR005905 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
2dln - VANCOMYCIN RESISTANCE: STRUCTURE OF D-ALANINE:D-ALANINE LIGASE AT 2.3 ANGSTROMS RESOLUTION (2.3 Å) PDBe PDBsum 2dln
Catalytic CATH Domains
3.30.1490.20 CATHdb 3.40.50.20 CATHdb 3.30.470.20 CATHdb (see all for 2dln)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:6.3.2.4)

D-alanine zwitterion
CHEBI:57416ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
D-alanyl-D-alanine zwitterion
CHEBI:57822ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: MurE synthetase, Alanine:alanine ligase (ADP-forming), Alanylalanine synthetase, D-Ala-D-Ala synthetase, D-alanylalanine synthetase, D-alanyl-D-alanine synthetase,

Enzyme Mechanism

Introduction

This enzyme catalyses the transfer of a phosphate group from ATP to carboxyl oxygen of D-alanine, forming an intermediate, D-alanyl acylphosphate. Then the acyl group is transferred from D-alanyl acylphosphate to the amine group of the second D-alanine substrate, releasing inorganic phosphate.

Catalytic Residues Roles

UniProt PDB* (2dln)
Asn272 Asn272A Forms part of the magnesium 2 binding site. metal ligand
Ser150 Ser150A Helps stabilise the acyl-phosphate intermediates and transition states. Also accepts a hydrogen bond from Tyr216, allowing that residue to activate the second substrate amino acid. electrostatic stabiliser
Arg255 Arg255A Has a dual function: (1) substrate orientation in subsite 2, (2) stabilisation of the transition state as part of the oxyanion hole in subsite 2. steric locator, electrostatic stabiliser
Asp257 Asp257A Forms part of the magnesium 1 binding site. metal ligand
Glu270 Glu270A Forms part of the binding site of both catalytic magnesium ions. metal ligand
Glu15 Glu15A Binds the amine group of the first substrate amino acid, helps guide the initial phosphate transfer and stabilise the transition state. steric locator, electrostatic stabiliser
Gly276 (main-N) Gly276A (main-N) Forms part of the oxyanion hole. electrostatic stabiliser
Tyr216 Tyr216A The residue at this position may determine the activation energy and whether D-alanine or D-lactate is attached to D-alanyl acylphosphate. It is currently unclear if this residue acts as a general acid/base, or perturbates the pKa of the second substrate amino acid such that it binds in the neutral state. activator
*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

References

  1. Fan C et al. (1994), Science, 266, 439-443. Vancomycin resistance: structure of D-alanine:D-alanine ligase at 2.3 A resolution. PMID:7939684.
  2. Prosser GA et al. (2013), FEBS J, 280, 1150-1166. Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine. DOI:10.1111/febs.12108. PMID:23286234.
  3. Neuhaus FC (2010), J Mol Graph Model, 28, 728-734. Role of Arg301 in substrate orientation and catalysis in subsite 2 of D-alanine:D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: a molecular docking study. DOI:10.1016/j.jmgm.2010.01.010. PMID:20167520.
  4. Kitamura Y et al. (2009), Acta Crystallogr D Biol Crystallogr, 65, 1098-1106. Structure of D-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme-ligand interactions. DOI:10.1107/S0907444909029710. PMID:19770507.
  5. Lee JH et al. (2006), Proteins, 64, 1078-1082. Crystal structure of the apo form of D-alanine: D-alanine ligase (Ddl) from Thermus caldophilus: a basis for the substrate-induced conformational changes. DOI:10.1002/prot.20927. PMID:16779845.
  6. van Heijenoort J (2001), Nat Prod Rep, 18, 503-519. Recent advances in the formation of the bacterial peptidoglycan monomer unit. PMID:11699883.
  7. Kuzin AP et al. (2000), Structure, 8, 463-470. Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides. PMID:10801495.
  8. Fan C et al. (1997), Biochemistry, 36, 2531-2538. D-alanine:D-alanine ligase: phosphonate and phosphinate intermediates with wild type and the Y216F mutant. DOI:10.1021/bi962431t. PMID:9054558.
  9. Park IS et al. (1997), J Biol Chem, 272, 9210-9214. D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation. PMID:9083053.
  10. Park IS et al. (1996), Biochemistry, 35, 10464-10471. Gain of D-alanyl-D-lactate or D-lactyl-D-alanine synthetase activities in three active-site mutants of the Escherichia coli D-alanyl-D-alanine ligase B. DOI:10.1021/bi9603128. PMID:8756703.
  11. Shi Y et al. (1995), Biochemistry, 34, 2768-2776. Active site mapping of Escherichia coli D-Ala-D-Ala ligase by structure-based mutagenesis. PMID:7893688.
  12. Fan C et al. (1995), Proc Natl Acad Sci U S A, 92, 1172-1176. A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli. PMID:7862655.
  13. McDermott AE et al. (1990), Biochemistry, 29, 5767-5775. Rotational resonance determination of the structure of an enzyme-inhibitor complex: phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP. PMID:2200515.
  14. Greenlee WJ et al. (1989), J Med Chem, 32, 165-170. Synthesis of an analogue of tabtoxinine as a potential inhibitor of D-alanine:D-alanine ligase (ADP forming). PMID:2562853.
  15. Duncan K et al. (1988), Biochemistry, 27, 3709-3714. ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium D-alanine:D-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of D-alanine. PMID:3044448.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg255A electrostatic stabiliser
Gly276A (main-N) electrostatic stabiliser
Asp257A metal ligand
Glu270A metal ligand
Asn272A metal ligand
Tyr216A activator
Arg255A steric locator
Glu15A electrostatic stabiliser, steric locator
Ser150A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Carine Berezin, Craig Porter, Gemma L. Holliday