Threonine---tRNA ligase

 

threonyl tRNA synthase from E.coli is able to catalyse the attachment of a threonine residue to its corresponding tRNA, displaying remarkable specificity as it only works on the specific amino acid and on the specific tRNA. It is part of the class II tRNA synthases, which make up approximately half of the known tRNA synthases. It is rare among class II synthases because of the presence of a Zinc ion in the active site, which is catalytic rather than structural.

 

Reference Protein and Structure

Sequence
P0A8M3 UniProt (6.1.1.3) IPR002320 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1qf6 - STRUCTURE OF E. COLI THREONYL-TRNA SYNTHETASE COMPLEXED WITH ITS COGNATE TRNA (2.9 Å) PDBe PDBsum 1qf6
Catalytic CATH Domains
3.30.930.10 CATHdb (see all for 1qf6)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:6.1.1.3)

AMP 3'-end(1-) residue
CHEBI:78442ChEBI
+
L-threonine zwitterion
CHEBI:57926ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
hydron
CHEBI:15378ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
+
3'-(L-threonyl)adenylyl(1-) group
CHEBI:78534ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
Alternative enzyme names: TRS, Threonine translase, Threonine-transfer ribonucleate synthetase, Threonyl ribonucleic synthetase, Threonyl-tRNA synthetase, Threonyl-transfer RNA synthetase, Threonyl-transfer ribonucleate synthetase, Threonyl-transfer ribonucleic acid synthetase,

Enzyme Mechanism

Introduction

The reaction proceeds through direct nucleophilic attack from the carboxy terminal of the threonine on the alpha phosphate of ATP to form threonylAMP and releasing PPi in the process. This part of the reaction passes through a pentavalent phosphate intermediate, stabilised by Arg 363's positively charged side chain. The threonylAMP binds specifically to the Zinc ion, which therefore prevents other amino acids from forming amino acylAMPs. Placement of the tRNA anticodon allows specific transfer of the threonyl moiety to it by another nucleophilic addition elimination reaction, this time with AMP as the leaving group.

Catalytic Residues Roles

UniProt PDB* (1qf6)
Lys465, Gln381 Lys465A(B), Gln381A(B) The residues are thought to help stabilise negative charge during the reaction. electrostatic stabiliser
Arg363 Arg363A(B) Positive charge on side chain interacts electrostatically with the pentavalent phosphate intermediate thus resulting in its stabilisation, facilitating the formation of the aminoacyl AMP. electrostatic stabiliser
Cys334, His385, His511 Cys334A(B), His385A(B), His511A(B) Forms part of the catalytic zinc binding site. metal ligand, 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, intermediate formation, overall reactant used, proton transfer, intermediate collapse, overall product formed

References

  1. Sankaranarayanan R et al. (1999), Cell, 97, 371-381. The Structure of Threonyl-tRNA Synthetase-tRNAThr Complex Enlightens Its Repressor Activity and Reveals an Essential Zinc Ion in the Active Site. DOI:10.1016/s0092-8674(00)80746-1. PMID:10319817.
  2. Aboelnga MM et al. (2017), J Phys Chem B, 121, 6163-6174. Roles of the Active Site Zn(II) and Residues in Substrate Discrimination by Threonyl-tRNA Synthetase: An MD and QM/MM Investigation. DOI:10.1021/acs.jpcb.7b03782. PMID:28592109.
  3. Perona JJ et al. (2014), Top Curr Chem, 344, 1-41. Synthetic and editing mechanisms of aminoacyl-tRNA synthetases. DOI:10.1007/128_2013_456. PMID:23852030.
  4. Torres-Larios A et al. (2003), J Mol Biol, 331, 201-211. Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase. PMID:12875846.
  5. Musier-Forsyth K et al. (2000), Nat Struct Biol, 7, 435-436. Role of zinc ion in translational accuracy becomes crystal clear. DOI:10.1038/75816. PMID:10881182.
  6. Sankaranarayanan R et al. (2000), Nat Struct Biol, 7, 461-465. Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase. DOI:10.1038/75856. PMID:10881191.

Step 1. The first step of the reaction involves nucleophilic attack from the carboxy terminal of the threonine on the alpha phosphate of the ATP. This forms threonylAMP and releases PPi. The reaction is stabilised by the positively charged Arg363 side chain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys334A(B) metal ligand
His385A(B) metal ligand
His511A(B) metal ligand
Arg363A(B) electrostatic stabiliser
Gln381A(B) electrostatic stabiliser
Asp383A(B) electrostatic stabiliser
Lys465A(B) electrostatic stabiliser

Chemical Components

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

Step 2. The amine group of threonylAMP deprotonates the 3'-OH group of the tRNA. This is followed by nucleophilic attack of the oxygen on the carbonyl group of the threoylAMP, eliminating AMP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys334A(B) electrostatic stabiliser
Arg363A(B) electrostatic stabiliser
Gln381A(B) electrostatic stabiliser
Asp383A(B) electrostatic stabiliser
Cys334A(B) metal ligand
His511A(B) metal ligand
His385A(B) metal ligand

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, intermediate collapse, overall product formed
Download: Image, Marvin File

Step 1. The first step of the reaction involves nucleophilic attack from the carboxy terminal of the threonine on the alpha phosphate of the ATP. This forms threonylAMP and releases PPi. The reaction is stabilised by the positively charged Arg363 side chain.

Step 2. The amine group of threonylAMP deprotonates the 3'-OH group of the tRNA. This is followed by nucleophilic attack of the oxygen on the carbonyl group of the threoylAMP, eliminating AMP.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Amelia Brasnett