Rational design to block amino acid editing of a tRNA synthetase

Investigations in chemical biology have focused on the synthesis of custom-designed proteins with site-specific incorporation of novel amino acids. Their success requires stable production of misacylated tRNAs. Utilization of aminoacyl-tRNA synthetases has been hindered because of enzyme molecular recognition mechanisms that ensure high fidelity of protein synthesis. Leucyl-tRNA synthetase naturally misaminoacylates chemically diverse standard and nonstandard amino acids, but contains a separate amino acid editing active site to hydrolyze incorrectly mischarged tRNAs. In this work, a rational mutagenesis design to block enzyme editing is described and involves substitution of bulky amino acids into the amino acid binding pocket of the hydrolytic active site. These engineered enzymes stably misacylated isoleucine to tRNALeu. The use of these mutant leucyl-tRNA synthetases has the potential to produce pools of mischarged tRNAs that are linked to nonstandard amino acids for in vitro translation. In addition,...