(carboxyethyl)arginine beta-lactam-synthase

 

Asparagine synthetase B catalyses the ATP-dependent conversion of aspartate to asparagine [PMID: 11551215]. This enzyme is a homodimer, with each monomer composed of a glutaminase domain and a synthetase domain. It forms part of the pathway for the biosythesis of the beta-lactamase inhibitor clavulanate in Streptomyces clavuligerus. This entry represents the reaction occuring in the synthetase domain in which the L-N2-(2-carboxyethyl)arginine is first converted into an acyl-AMP by reaction with ATP and loss of diphosphate, and that the beta-lactam ring is then formed by the intramolecular attack of the beta-nitrogen on the activated carboxy group [PMID:9689037].

 

Reference Protein and Structure

Sequence
P0DJQ7 UniProt (6.3.3.4) IPR001962 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces clavuligerus (Bacteria) Uniprot
PDB
1mb9 - BETA-LACTAM SYNTHETASE COMPLEXED WITH ATP (2.11 Å) PDBe PDBsum 1mb9
Catalytic CATH Domains
3.40.50.620 CATHdb (see all for 1mb9)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.3.4)

ATP(4-)
CHEBI:30616ChEBI
+
N(2)-(2-carboxyethyl)-L-arginine dizwitterion
CHEBI:57304ChEBI
→
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
deoxyamidinoproclavaminic acid zwitterion
CHEBI:57303ChEBI
+
hydron
CHEBI:15378ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
Alternative enzyme names: Beta-lactam synthetase, L-2-N-(2-carboxyethyl)arginine cyclo-ligase (AMP-forming),

Enzyme Mechanism

Introduction

The L-N2-(2-carboxyethyl)arginine substrate initiates a nucleophilic attack on the alpha-phosphate of ATP in a substitution reaction. The beta-lactam ring is then formed by the intramolecular attack of the beta-nitrogen on the activated carboxy group. It is worth noting that in this protein the Gly-Tyr dyad is actually in the reverse of the "normal" protonation state, i.e. Glu is neutral and Tyr is negatively charged [PMID:19371088].

Catalytic Residues Roles

UniProt PDB* (1mb9)
Glu382 Glu382A This residue is neutral in the enzyme's ground state. Helps activate the catalytic tyrosine. increase basicity, hydrogen bond donor, electrostatic stabiliser, increase acidity
Tyr348 Tyr348A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys443 Lys443A Helps stabilise the reactive intermediates formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
*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, proton transfer, intermediate collapse, cyclisation, inferred reaction step, native state of enzyme regenerated

References

  1. Miller MT et al. (2002), Proc Natl Acad Sci U S A, 99, 14752-14757. The catalytic cycle of  -lactam synthetase observed by x-ray crystallographic snapshots. DOI:10.1073/pnas.232361199. PMID:12409610.
  2. Labonte JW et al. (2012), Medchemcomm, 3, 960-966. Engineering the synthetic potential of β-lactam synthetase and the importance of catalytic loop dynamics. DOI:10.1039/c2md00305h. PMID:23616913.
  3. Raber ML et al. (2009), Chembiochem, 10, 2904-2912. A Conserved Lysine in β-Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis. DOI:10.1002/cbic.200900389. PMID:19882698.
  4. Raber ML et al. (2009), Biochemistry, 48, 4959-4971. A Conserved Tyrosyl−Glutamyl Catalytic Dyad in Evolutionarily Linked Enzymes: Carbapenam Synthetase and β-Lactam Synthetase. DOI:10.1021/bi900432n. PMID:19371088.

Step 1. The L-N2-(2-carboxyethyl)arginine substrate initiates a nucleophilic attack on the alphs-phosphate of ATP in a substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr348A hydrogen bond acceptor
Glu382A hydrogen bond donor, electrostatic stabiliser
Lys443A hydrogen bond donor

Chemical Components

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

Step 2. Tyr348 deprotonates the immine, which initiates a nucleophilic attack on the carbonyl carbon next to the phosphate group in a substitution reaction. The leaving phosphate group deprotonates the newly formed beta-lactam. It is thought that this is a concerted mechanism which proceeds via a tetrahedral transition state, instead of the stabilised intermediate [PMID:12409610].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr348A hydrogen bond acceptor
Glu382A hydrogen bond donor, increase basicity, electrostatic stabiliser
Lys443A hydrogen bond donor, electrostatic stabiliser
Tyr348A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall product formed, intermediate collapse, cyclisation

Step 3. Water deprotonates Tyr348 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr348A hydrogen bond donor
Glu382A hydrogen bond donor, increase acidity, electrostatic stabiliser
Tyr348A proton donor

Chemical Components

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

Step 1. The L-N2-(2-carboxyethyl)arginine substrate initiates a nucleophilic attack on the alphs-phosphate of ATP in a substitution reaction.

Step 2. Tyr348 deprotonates the immine, which initiates a nucleophilic attack on the carbonyl carbon next to the phosphate group in a substitution reaction. The leaving phosphate group deprotonates the newly formed beta-lactam. It is thought that this is a concerted mechanism which proceeds via a tetrahedral transition state, instead of the stabilised intermediate [PMID:12409610].

Step 3. Water deprotonates Tyr348 in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Charity Hornby