UDP-N-acetylmuramoyl-L-alanine---D-glutamate ligase

 

UDP-N-acetylmuramoylalanine-D-glutamate ligase catalyses the formation of the peptide bond between UDP-N-acetylmuramoyl-L-alanine and D-glutamate in peptidoglycan biosynthesis. Peptidoglycan is formed as linear repeating disaccharide chains interconnected by a short peptide moiety. It is found in bacterial cell walls and hence peptidoglycan biosynthetic enzymes are possible targets for antibacterial drugs.

 

Reference Protein and Structure

Sequence
P14900 UniProt (6.3.2.9) IPR005762 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1uag - UDP-N-ACETYLMURAMOYL-L-ALANINE:D-GLUTAMATE LIGASE (1.95 Å) PDBe PDBsum 1uag
Catalytic CATH Domains
3.40.1190.10 CATHdb (see all for 1uag)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.2.9)

D-glutamate(1-)
CHEBI:29986ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
UDP-N-acetyl-alpha-D-muramoyl-L-alaninate(3-)
CHEBI:83898ChEBI
→
hydron
CHEBI:15378ChEBI
+
UDP-N-acetyl-alpha-D-muramoyl-L-alanyl-D-glutamate(4-)
CHEBI:83900ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
Alternative enzyme names: D-glutamate ligase, D-glutamate-adding enzyme, MurD synthetase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase, UDP-Mur-NAC-L-Ala:D-Glu ligase, Uridine diphospho-N-acetylmuramoylalanyl-D-glutamate synthetase, UDP-N-acetylmuramoyl-L-alanine:glutamate ligase (ADP-forming), UDP-N-acetylmuramoylalanine--D-glutamate ligase, UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (ADP-forming),

Enzyme Mechanism

Introduction

The reaction can be broken down into three steps. The first is the initial phosphorylation of the UMA carboxylate to form the acyl-phosphate. The intermediate would be stabilised by Lys115 and the two divalent cations in the active site. The second is the nucleophilic attack by the amine of D-glutamate producing a tetrahedral intermediate. The formation of the tetrahedral adduct requires a catalytic base to abstract the proton from the D-glutamate amine. This is achieved by either D-glutamate binding in the from solution in the free base form or the deprotonation is assisted by the gamma phosphate of ATP. In the last step the tetrahedral intermediate collapses to yield UDP-N-acetylmuramoyl-L-alanyl-D-glutamate, ADP and inorganic phosphate. This requires another catalytic base to remove the second proton. His183 is thought to act as such a base.

Catalytic Residues Roles

UniProt PDB* (1uag)
His184 His183A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, steric role
Lys116, Asn139 Lys115A, Asn138A Acts to stabilise the reactive intermediates and transition states formed during the course of the reaction. attractive charge-charge interaction, hydrogen bond donor, activator, electrostatic stabiliser, increase electrophilicity
*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, overall product formed, intermediate formation, proton transfer, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, inferred reaction step, native state of enzyme regenerated

References

  1. Bertrand JA et al. (1999), J Mol Biol, 289, 579-590. Determination of the MurD mechanism through crystallographic analysis of enzyme complexes. DOI:10.1006/jmbi.1999.2800. PMID:10356330.
  2. Šink R et al. (2016), PLoS One, 11, e0152075-. Crystallographic Study of Peptidoglycan Biosynthesis Enzyme MurD: Domain Movement Revisited. DOI:10.1371/journal.pone.0152075. PMID:27031227.
  3. Perdih A et al. (2009), Proteins, 74, 744-759. MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method. DOI:10.1002/prot.22188. PMID:18704940.
  4. Bertrand JA et al. (1997), EMBO J, 16, 3416-3425. Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli. DOI:10.1093/emboj/16.12.3416. PMID:9218784.

Step 1. The carboxylate group of UDP-N-acetylmuramoyl-L-alanine attacks the gamma phosphate of ATP, forming an activated intermediate and ADP.

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Catalytic Residues Roles

Residue Roles
Lys115A attractive charge-charge interaction, electrostatic stabiliser, increase electrophilicity, activator
Asn138A electrostatic stabiliser, hydrogen bond donor, steric role
His183A hydrogen bond donor, steric role

Chemical Components

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

Step 2. The terminal phosphate of the departing ADP abstracts a proton from the amine group of glutamine. The amine group of glutamate then attacks the activated phospho-carbonyl, forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Asn138A hydrogen bond donor, steric role, electrostatic stabiliser
His183A hydrogen bond donor

Chemical Components

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

Step 3. His183A deprotonates the intermediate.

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Catalytic Residues Roles

Residue Roles
Asn138A hydrogen bond donor, electrostatic stabiliser
His183A hydrogen bond acceptor, hydrogen bond donor
His183A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The tetrahedral intermediate collapses, releasing a peptidoglycan molecule and protonated phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys115A hydrogen bond donor
Asn138A electrostatic stabiliser, hydrogen bond donor
His183A hydrogen bond donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, overall product formed

Step 5. His183A is deprotonated by a solvent molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His183A hydrogen bond donor, proton donor

Chemical Components

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

Step 1. The carboxylate group of UDP-N-acetylmuramoyl-L-alanine attacks the gamma phosphate of ATP, forming an activated intermediate and ADP.

Step 2. The terminal phosphate of the departing ADP abstracts a proton from the amine group of glutamine. The amine group of glutamate then attacks the activated phospho-carbonyl, forming a tetrahedral intermediate.

Step 3. His183A deprotonates the intermediate.

Step 4. The tetrahedral intermediate collapses, releasing a peptidoglycan molecule and protonated phosphate.

Step 5. His183A is deprotonated by a solvent molecule.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Christian Drew, Craig Porter, Charity Hornby