Threonine synthase

 

Threonine synthase (TS) from Saccharomyces cerevisiae catalyses the conversion of O-phospho-L-homoserine (OPHS) into threonine and phosphate. This is the final step in threonine biosynthesis. The enzyme requires a pyridoxal 5'-phosphate (PLP) cofactor which binds at the interface of all three of the protein's domains.

 

Reference Protein and Structure

Sequence
P16120 UniProt (4.2.3.1) IPR004450 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1kl7 - Crystal Structure of Threonine Synthase from Yeast (2.7 Å) PDBe PDBsum 1kl7
Catalytic CATH Domains
3.40.50.1100 CATHdb (see all for 1kl7)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
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Enzyme Reaction (EC:4.2.3.1)

water
CHEBI:15377ChEBI
+
O-phosphonato-L-homoserine(2-)
CHEBI:57590ChEBI
→
L-threonine zwitterion
CHEBI:57926ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: Threonine synthetase, O-phospho-L-homoserine phospho-lyase (adding water),

Enzyme Mechanism

Introduction

The catalytic cycle starts with a transaldimination reaction. Lys124 which is initially bound to the PLP cofactor is replaced by OPHS forming the external aldimine. Lys124 catalyses the abstraction of the C-alpha proton of the substrate and its transfer to the PLP C4' position. Next, Lys124 stereospecifically abstracts the beta-pro-S hydrogen leading to the non-hydrolytic elimination of the gamma-phosphate. The gamma-methylene group left after the elimination is reprotonated producing the PLP-derivative of E-aminocrotonate. Next, water is added at C-beta. Finally, reverse transaldimination yields L-threonine.

Catalytic Residues Roles

UniProt PDB* (1kl7)
Lys124 Lys124A Lys124 is the only residue in the immediate environment of the bound substrate that can act as an acid-base catalyst. It is involved in the abstraction of a proton from C-alpha and it's transfer to C4' as well as the abstraction of a proton from C-beta and the protonation of the C-gamma in the methylene intermediate. In the last step, Lys124 acts as a nucleophile attacking the external aldimine resulting in the release of the L-threonine. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, proton relay, increase nucleophilicity, electron pair acceptor, electron pair 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, intermediate formation, cofactor used, overall reactant used, intramolecular elimination, schiff base formed, intermediate collapse, proton relay, assisted tautomerisation (not keto-enol), proton transfer, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, intermediate terminated

References

  1. Garrido-Franco M et al. (2002), J Biol Chem, 277, 12396-12405. Structure and Function of Threonine Synthase from Yeast. DOI:10.1074/jbc.m108734200. PMID:11756443.
  2. Ujiie Y et al. (2017), J Phys Chem B, 121, 5536-5543. Molecular Mechanism of the Reaction Specificity in Threonine Synthase: Importance of the Substrate Conformations. DOI:10.1021/acs.jpcb.7b02932. PMID:28489381.

Step 1. The amino group of the serine performs a nucleophilic attack on the imine carbon of the lys-bound PLP forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Lys124A covalently attached
Lys124A electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, cofactor used, overall reactant used

Step 2. Lys124 is cleaved from PLP leaving the substrate bound.

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Catalytic Residues Roles

Residue Roles
Lys124A nucleofuge

Chemical Components

ingold: intramolecular elimination, schiff base formed, intermediate collapse

Step 3. Lys124 abstracts a proton from the alpha carbon of the substrate causing tautomerization of the imine.

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Catalytic Residues Roles

Residue Roles
Lys124A proton relay
Lys124A proton acceptor, proton donor

Chemical Components

proton relay, assisted tautomerisation (not keto-enol)

Step 4. Lys124 abstracts a proton from the beta carbon causing a second tautomerization from imine to enamine.

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Catalytic Residues Roles

Residue Roles
Lys124A proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol)

Step 5. Phosphate is eliminated from the homoserine and another imine is formed.

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Catalytic Residues Roles

Residue Roles
Lys124A proton donor

Chemical Components

schiff base formed, ingold: intramolecular elimination, proton transfer, assisted tautomerisation (not keto-enol)

Step 6. Lys124 acts as a proton relay causing another tautomerization reaction to occur.

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Catalytic Residues Roles

Residue Roles
Lys124A proton relay, proton donor, proton acceptor

Chemical Components

proton relay, assisted tautomerisation (not keto-enol)

Step 7. Water performs a nucleophilic attack on the C=C bond of the substrate, forming a carboanion. The water is activated by Lys124.

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Catalytic Residues Roles

Residue Roles
Lys124A increase nucleophilicity, proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition

Step 8. The carboanion abstracts a proton from Lys124.

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Catalytic Residues Roles

Residue Roles
Lys124A proton donor

Chemical Components

proton transfer

Step 9. The amino group of Lys124 performs a nucleophilic attack on the imine carbon of the thr-bound PLP forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Lys124A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 10. Threonine is cleaved from PLP leaving Lys124 bound.

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Catalytic Residues Roles

Residue Roles
Lys124A electron pair donor

Chemical Components

schiff base formed, ingold: intramolecular elimination, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, intermediate terminated
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Step 1. The amino group of the serine performs a nucleophilic attack on the imine carbon of the lys-bound PLP forming a tetrahedral intermediate.

Step 2. Lys124 is cleaved from PLP leaving the substrate bound.

Step 3. Lys124 abstracts a proton from the alpha carbon of the substrate causing tautomerization of the imine.

Step 4. Lys124 abstracts a proton from the beta carbon causing a second tautomerization from imine to enamine.

Step 5. Phosphate is eliminated from the homoserine and another imine is formed.

Step 6. Lys124 acts as a proton relay causing another tautomerization reaction to occur.

Step 7. Water performs a nucleophilic attack on the C=C bond of the substrate, forming a carboanion. The water is activated by Lys124.

Step 8. The carboanion abstracts a proton from Lys124.

Step 9. The amino group of Lys124 performs a nucleophilic attack on the imine carbon of the thr-bound PLP forming a tetrahedral intermediate.

Step 10. Threonine is cleaved from PLP leaving Lys124 bound.

Products.

Contributors

Gemma L. Holliday, James Willey