PDBsum entry 5onh

Ligase

PDB id: 5onh

Contents

Protein chains

860 a.a.

DNA/RNA

Ligands

LSS ×2

VRT ×2

Metals

_MG ×2

_ZN ×2

PDB id:

5onh

Name:

Ligase

Title:

Quaternary complex of wild type e. Coli leucyl-tRNA synthetase with tRNA(leu), leucyl-adenylate analogue, and post-transfer editing analogue of norvaline in the aminoacylation conformation

Structure:

Leucine--tRNA ligase. Chain: a, d. Synonym: leucyl-tRNA synthetase,leurs. Engineered: yes. L-leucyl-tRNA(leu). Chain: b, e. Engineered: yes

Source:

Escherichia coli k-12. Organism_taxid: 83333. Gene: leus, b0642, jw0637. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Synthetic: yes. Escherichia coli. Organism_taxid: 562

Resolution:

3.10Å R-factor: 0.224 R-free: 0.257

Authors:

A.Palencia,S.Cusack

Key ref:

M.Dulic et al. (2018). Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase. J Mol Biol, 430, 1. PubMed id: 29111343 DOI: 10.1016/j.jmb.2017.10.024

Date:

03-Aug-17 Release date: 15-Nov-17

Protein chains

P07813  (SYL_ECOLI) -  Leucine--tRNA ligase from Escherichia coli (strain K12)

Seq: 860 a.a.

Struc: 860 a.a.

DNA/RNA chains

G-C-C-C-G-G-A-U-G-G-U-G-G-A-A-U-C-G-G-U-A-G-A-C-A-C-A-A-G-G-G-A-U-U-A-A-U-C-C- 82 bases

G-C-C-C-G-G-A-U-G-G-U-G-G-A-A-U-C-G-G-U-A-G-A-C-A-C-A-A-G-G-G-A-U-A-A-U-C-C-C- 83 bases

Enzyme reactions

• Enzyme class: E.C.6.1.1.4 - leucine--tRNA ligase.
• Reaction: tRNA(Leu) + L-leucine + ATP = L-leucyl-tRNA(Leu) + AMP + diphosphate

tRNA(Leu) + L-leucine + ATP = L-leucyl-tRNA(Leu) (Bound ligand (Het Group name = LSS) matches with 54.29% similarity) + AMP + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2017.10.024

J Mol Biol 430:1 (2018)

PubMed id: 29111343

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase.

M.Dulic, N.Cvetesic, I.Zivkovic, A.Palencia, S.Cusack, B.Bertosa, I.Gruic-Sovulj.

ABSTRACT

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA^Leuand their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA^Leudiffered by about 10⁴-fold. We further established the critical role for the A76 3'-OH group of the tRNA^Leuin post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA^Leuhydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.