title: Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism.
abstract: The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Zhai
authors.forename: Susan A
authors.initials: SA
authors.lastname: Martinis
mesh_terms: D000215:Acylation; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry*; D009154:Mutation; D017393:RNA Editing; D013816:Thermodynamics; D013912:Threonine
chemical_list: D029968:Escherichia coli Proteins; D013912:Threonine; D007935:Leucine-tRNA Ligase