Dihydrodipicolinate synthase

 

Dihydrodipicolinate synthase (DHDPS) catalyses the aldol-like condensation of pyruvate and L-aspartate beta semi-aldehyde (S-ASA). It is an enzyme involved in the lysine biosynthetic pathway of procaryotes, some Phycomycetes and higher plants. Since this pathway is not present in animals, pathway members such as DHDPS have become attractive targets for rational antibiotic and herbicide drug design.

 

Reference Protein and Structure

Sequence
P0A6L2 UniProt (4.3.3.7) IPR005263 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1dhp - DIHYDRODIPICOLINATE SYNTHASE (2.3 Å) PDBe PDBsum 1dhp
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1dhp)
Click To Show Structure

Enzyme Reaction (EC:4.3.3.7)

pyruvate
CHEBI:15361ChEBI
+
L-aspartic acid 4-semialdehyde betaine
CHEBI:537519ChEBI
→
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate(2-)
CHEBI:67139ChEBI
+
hydron
CHEBI:15378ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: Dihydrodipicolinic acid synthase, Dihydropicolinate synthetase, Dihydrodipicolinate synthetase, DHDPS, L-aspartate-4-semialdehyde hydro-lyase (adding pyruvate and cyclizing), DapA (gene name),

Enzyme Mechanism

Introduction

In the first step, the E-amino group of the active site Lys161 acts as a nucleophile towards the keto group of pyruvate, forming an internal Schiff base adduct. Tyr133 is aligned to form a hydrogen bond with the keto oxygen of pyruvate which accelerates the dehydration of the tetrahedral intermediate during Schiff base formation. The enamine tautomer of the Schiff base adds to the dehydrated (S)-ASA, and this species then undergoes cyclisation to form HTPA. This cyclisation could occur outside of the active site, with water acting as the nucleophile and displacing Lys161 from the Schiff base adduct. A conserved strained peptide carbonyl is thought to enhance the reactivity of the active site by polarizing the pyruvate substrate. Also, the release of strain in the peptide conformation, triggered by the binding of (S)-ASA may couple to the condensation reaction and increase the free energy associated with catalysis.

Catalytic Residues Roles

UniProt PDB* (1dhp)
Arg138 Arg138A Acts as an electrostatic stabilizer for the carboxyl group. electrostatic stabiliser
Ile203 (main-C) Ile203A (main-C) The backbone carbonyl adopts a strained conformation within the active site. The binding of (S)-SAS releases the carbonyl, and so increases the free energy available to the reaction. The carbonyl is also thought to polarise the pyruvate substrate towards attack by Lys161. increase electrophilicity, activator, polar interaction, steric role
Tyr133 Tyr133A The residue's phenolic oxygen forms a hydrogen bond to the reacting pyruvate carbonyl, activating the group towards nucleophilic attack by Ly161. It is also thought to encourage the collapse of the tetrahedral intermediate during Schiff base formation. hydrogen bond donor, proton acceptor, proton donor, proton relay, activator, electrostatic stabiliser
Lys161 Lys161A The side chain amine group acts as a nucleophile towards the pyruvate keto functionality, forming a Schiff base linkage. The linkage is broken either in the active site by intramolecular 6-exo-tet cyclisation followed by elimination or outside of the active site by hydrolysis followed by cyclisation. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, electron pair acceptor, electron pair donor
Thr44, Tyr107 Thr44A, Tyr107B Part of the proton relay with Tyr133 hydrogen bond acceptor, hydrogen bond donor
*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 addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton transfer, inferred reaction step, unimolecular elimination by the conjugate base, tautomerisation (not keto-enol), dehydration, intramolecular nucleophilic addition, cyclisation, proton relay, enzyme-substrate complex cleavage, native state of enzyme regenerated, overall product formed, intermediate terminated

References

  1. Blickling S et al. (1997), Biochemistry, 36, 24-33. Reaction Mechanism ofEscherichia coliDihydrodipicolinate Synthase Investigated by X-ray Crystallography and NMR Spectroscopy†,‡. DOI:10.1021/bi962272d. PMID:8993314.
  2. Dobson RC et al. (2009), Biochimie, 91, 1036-1044. Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis. DOI:doi:10.1016/j.biochi.2009.05.013.
  3. Dobson RC et al. (2008), Protein Sci, 17, 2080-2090. Conserved main-chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase. DOI:10.1110/ps.037440.108. PMID:18787203.

Step 1. Lys161 acts as a nucleophile towards the keto group of the pyruvate substrate, forming a covalently bound enzyme-substrate intermediate. The pyruvate is polarised by the presence of the main chain carbonyl of Ile203, increasing its electrophilicity.

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Catalytic Residues Roles

Residue Roles
Ile203A (main-C) polar interaction, increase electrophilicity
Tyr133A hydrogen bond donor, electrostatic stabiliser
Arg138A electrostatic stabiliser
Lys161A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 2. Proton transfer activates Schiff base formation in the next reaction step.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached, hydrogen bond donor
Tyr133A electrostatic stabiliser, hydrogen bond donor
Arg138A electrostatic stabiliser
Lys161A proton donor

Chemical Components

proton transfer, inferred reaction step, intermediate formation

Step 3. Elimination of water results in Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached, hydrogen bond donor
Tyr133A activator, hydrogen bond donor
Arg138A electrostatic stabiliser
Lys161A proton donor, electron pair donor

Chemical Components

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

Step 4. The covalently bound lysine abstracts a proton from the terminal carbon to form the ene tautomer.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached, hydrogen bond acceptor
Arg138A electrostatic stabiliser
Lys161A proton acceptor, electron pair acceptor

Chemical Components

tautomerisation (not keto-enol), intermediate formation

Step 5. L-aspartate semi aldehyde is thought to enter the active site in the hydrate form. It then undergoes reversible dehydration to form the protonated aldehyde form.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached
Tyr133A activator, hydrogen bond donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, overall reactant used, dehydration, intermediate formation

Step 6. The enamine tautomer of the Schiff base adds to the dehydrated L-aspartate semi aldehyde in a conjugate nucleophilic attack.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached
Ile203A (main-C) steric role, increase electrophilicity
Arg138A electrostatic stabiliser
Lys161A electron pair donor

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation

Step 7. A 6-exo-tet cyclisation (Baldwin's classification) occurs in the enzyme-substrate intermediate. Dobson et al. [PMID:18787203] have suggested that the cyclisation process may occur outside of the active site and that once addition has occurred, hydrolysis breaks the enzyme-substrate bond and the acyclic intermediate leaves the active site. In this alternative mechanism, the hydrolytic water is activated by the carbonyl backbone of Ile203

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached
Ile203A (main-C) activator, steric role
Arg138A electrostatic stabiliser
Lys161A electron pair acceptor

Chemical Components

ingold: intramolecular nucleophilic addition, cyclisation, intermediate formation

Step 8. Proton transfer is mediated through Tyr133.

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Catalytic Residues Roles

Residue Roles
Lys161A covalently attached, hydrogen bond acceptor
Tyr133A proton relay
Arg138A electrostatic stabiliser
Thr44A hydrogen bond acceptor
Tyr107B hydrogen bond donor
Thr44A hydrogen bond donor
Tyr133A proton acceptor, proton donor
Lys161A proton acceptor

Chemical Components

proton transfer, proton relay, intermediate formation

Step 9. The product is formed and the active site is regenerated for further catalysis.

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Catalytic Residues Roles

Residue Roles
Tyr133A hydrogen bond donor
Arg138A electrostatic stabiliser
Lys161A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, native state of enzyme regenerated, overall product formed, intermediate terminated
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Step 1. Lys161 acts as a nucleophile towards the keto group of the pyruvate substrate, forming a covalently bound enzyme-substrate intermediate. The pyruvate is polarised by the presence of the main chain carbonyl of Ile203, increasing its electrophilicity.

Step 2. Proton transfer activates Schiff base formation in the next reaction step.

Step 3. Elimination of water results in Schiff base formation.

Step 4. The covalently bound lysine abstracts a proton from the terminal carbon to form the ene tautomer.

Step 5. L-aspartate semi aldehyde is thought to enter the active site in the hydrate form. It then undergoes reversible dehydration to form the protonated aldehyde form.

Step 6. The enamine tautomer of the Schiff base adds to the dehydrated L-aspartate semi aldehyde in a conjugate nucleophilic attack.

Step 7. A 6-exo-tet cyclisation (Baldwin's classification) occurs in the enzyme-substrate intermediate. Dobson et al. [PMID:18787203] have suggested that the cyclisation process may occur outside of the active site and that once addition has occurred, hydrolysis breaks the enzyme-substrate bond and the acyclic intermediate leaves the active site. In this alternative mechanism, the hydrolytic water is activated by the carbonyl backbone of Ile203

Step 8. Proton transfer is mediated through Tyr133.

Step 9. The product is formed and the active site is regenerated for further catalysis.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday, James W. Murray, Craig Porter, James Willey