title: Rational design to block amino acid editing of a tRNA synthetase.
abstract: 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, since many of the leucyl-tRNA synthetases do not interact with or rely upon the tRNA anticodon for identity, these enzymes may offer an excellent scaffold for the development of orthogonal tRNA synthetase/tRNA pairs that can potentially be used to customize protein synthesis.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Mursinna
authors.forename: Susan A
authors.initials: SA
authors.lastname: Martinis
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D007935:Leucine-tRNA Ligase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D007532:Isoleucine; D014443:Tyrosine; D010649:Phenylalanine; D008715:Methionine; D007935:Leucine-tRNA Ligase; D007930:Leucine