CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function, Biochemistry 39

ABSTRACT: The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a “coarse sieve”, while the reaction products of amino acids smaller than the cognate substrate will go through a “fine sieve ” and be hydrolyzed. This “double-sieve ” mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50 % of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNALeu by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNALeu made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function. Aminoacyl-tRNA synthetases (aaRSs)1 arose early in evolution and are believed to be a group of ancient enzymes that catalyze the precise charging of tRNAs with thei