N(2)-(2-carboxyethyl)arginine synthase

 

Thiamine-pyrophosphate dependent N(2)-(2-carboxyethyl)arginine synthase catalyses the initial step in the biosynthesis of the clinically relevant beta-lactamase inhibitor, clavulanic acid. The reaction involves the condensation of two primary metabolites, D-glyceraldehyde-3-phosphate and l-arginine to form N(2)-(2-carboxyethyl)arginine and phosphate. This C-N bond forming reaction forms an unusual alpha, beta-unsaturated acyl-thiamine-pyrophosphate intermediate. This enzyme is the first gene in the clavulanic acid cluster of the organism Streptomyces clavuligerus, with 18 other genes of the genome known to take part in this biosynthetic pathway. Besides this enzyme’s biosynthetic importance, it is also of mechanistic interest because of its unusual mechanism for a thiamine-pyrophosphate dependent enzyme.

As expected of a thiamine-pyrophosphate dependent enzyme, sequence analysis indicates the presence of a thiamine binding mode – a motif well conserved between enzymes which use this cofactor. The mechanism can require thiamine as a cofactor in two ways: i) to effect the nucleophilic attack at a carbonyl group, and ii) to stabilise the carbanion formed after proton transfer (or decarboxylation, as catalysed by some thiamine-pyrophosphate utilising enzymes). The enzyme is tetrameric, both physiologically and in crystallography data.

 

Reference Protein and Structure

Sequence
Q9LCV9 UniProt (2.5.1.66) IPR029061 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces clavuligerus (Bacteria) Uniprot
PDB
2iht - Carboxyethylarginine synthase from Streptomyces clavuligerus: SeMet structure (2.0 Å) PDBe PDBsum 2iht
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 2iht)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.66)

D-glyceraldehyde 3-phosphate(2-)
CHEBI:59776ChEBI
+
L-argininium(1+)
CHEBI:32682ChEBI
→
N(2)-(2-carboxyethyl)-L-arginine dizwitterion
CHEBI:57304ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: N(2)-(2-carboxyethyl)arginine synthetase, CEA synthetase, CEAS, Glyceraldehyde-3-phosphate:L-arginine 2-N-(2-hydroxy-3-oxopropyl) transferase (2-carboxyethyl-forming),

Enzyme Mechanism

Introduction

The active form of the thiamine pyrophosphate is formed on deprotonation by Glu45. The resulting stabilised, zwitterionic carbanion then attacks at the glyceraldehyde-3-phosphate aldehyde group, forming a cofactor-substrate adduct. In the absence of any residues available for general acid/base catalysis, the 4' amino group of the amino-pyridinium ring is proposed to play a key role in the mechanism. Like other TPP dependent enzymes, intramolecular proton transfer to the 4' amino group of this ring is known to activate the cofactor's reactive carbon centre, but the group is also thought to deprotonate the alpha carbon of the cofactor-substrate intermediate, initiating loss of water from the C2 position. The resulting enol is deprotonated which initiates an Ecb1 elimination of the phosphate group and the formation of a reactive 1,2 unsaturated-carbonyl, still tethered to the TPP cofactor. The L-arginine amine group then attacks at the double bond, which concomitant deprotonation of the nitrogen. Tautomerization and hydrolysis at the carbonyl releases the N(2)-(2-carboxyethyl)arginine product.

Catalytic Residues Roles

UniProt PDB* (2iht)
Tyr561 Tyr561A Positions a water which will coordinate to Mg2+ hydrogen bond acceptor, electrostatic stabiliser
Asp463, Asn490, Thr492 (main-C) Asp463A, Asn490A, Thr492A (main-C) Form Mg2+ binding site metal ligand
Phe438 Phe438A Phe438 orientates the thiamine pyrophosphate group in the active V conformation through steric interactions. steric role
Glu57 Glu57B Glu57 crucial for activation of cofactor. Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, 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

proton transfer, cofactor used, bimolecular nucleophilic addition, overall reactant used, intermediate formation, rate-determining step, unimolecular elimination by the conjugate base, elimination (not covered by the Ingold mechanisms), overall product formed, keto-enol tautomerisation, inferred reaction step, intermediate collapse, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Caines ME et al. (2009), Biochem Biophys Res Commun, 385, 512-517. Structural and mechanistic studies on N(2)-(2-carboxyethyl)arginine synthase. DOI:10.1016/j.bbrc.2009.05.095. PMID:19477162.
  2. 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.
  3. Caines ME et al. (2004), J Biol Chem, 279, 5685-5692. Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway. DOI:10.1074/jbc.M310803200. PMID:14623876.

Step 1. The active form of the thiamine pyrophosphate is formed on deprotonation by Glu45. Residue Phe438 orientates the thiamine pyrophosphate group in the active V conformation through steric interactions.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B activator, hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A electrostatic stabiliser, hydrogen bond acceptor
Glu57B proton acceptor

Chemical Components

proton transfer, cofactor used

Step 2. The stabilised, zwitterionic carbanion situated on the cofactor then attacks the glycer-aldehyde-3-phosphate carbonyl group, forming a cofactor-substrate adduct.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B activator, hydrogen bond donor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A electrostatic stabiliser, hydrogen bond acceptor
Glu57B proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, cofactor used, intermediate formation, rate-determining step

Step 3. The cofactor 4-amino group deprotonates the alpha carbon.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B activator, hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A electrostatic stabiliser, hydrogen bond acceptor
Glu57B proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The carbon anion eliminates water, forming an alkene in an E1cb mechanism. The departing water molecule abstracts a proton from the TPP subunit, which in turn abstracts a proton from Glu57.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B activator, hydrogen bond donor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A electrostatic stabiliser, hydrogen bond acceptor
Glu57B proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate formation

Step 5. The phosphate group is proposed to activate the enol towards 1,3 elimination of itself by acting as a base, releasing orthophosphate.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

proton transfer, elimination (not covered by the Ingold mechanisms), intermediate formation, overall product formed

Step 6. L-arginine attacks at the reactive alkene of the intermediate adduct, with concomitant protonation of the 1,3 ketone.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation

Step 7. The enol undergoes unassisted tautomerisation.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

keto-enol tautomerisation, intermediate formation, inferred reaction step

Step 8. Water attacks at the ketone carbonyl, initiating release of the carboxyethyl-arginine product.

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Catalytic Residues Roles

Residue Roles
Phe438A steric role
Glu57B hydrogen bond acceptor
Asp463A metal ligand
Asn490A metal ligand
Thr492A (main-C) metal ligand
Tyr561A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate collapse, native state of cofactor regenerated, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. The active form of the thiamine pyrophosphate is formed on deprotonation by Glu45. Residue Phe438 orientates the thiamine pyrophosphate group in the active V conformation through steric interactions.

Step 2. The stabilised, zwitterionic carbanion situated on the cofactor then attacks the glycer-aldehyde-3-phosphate carbonyl group, forming a cofactor-substrate adduct.

Step 3. The cofactor 4-amino group deprotonates the alpha carbon.

Step 4. The carbon anion eliminates water, forming an alkene in an E1cb mechanism. The departing water molecule abstracts a proton from the TPP subunit, which in turn abstracts a proton from Glu57.

Step 5. The phosphate group is proposed to activate the enol towards 1,3 elimination of itself by acting as a base, releasing orthophosphate.

Step 6. L-arginine attacks at the reactive alkene of the intermediate adduct, with concomitant protonation of the 1,3 ketone.

Step 7. The enol undergoes unassisted tautomerisation.

Step 8. Water attacks at the ketone carbonyl, initiating release of the carboxyethyl-arginine product.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, Charity Hornby