The prime function of Escherichia coli LeuRS CP1 domain is editing of norvaline not isoleucine

Leucyl-tRNA synthetase (LeuRS) is responsible for accurate charging of tRNALeu thus ensuring correct decoding of leucine codons. Using extensive steady-state and transient kinetic analyses we have recently characterized the capacity of Escherichia coli LeuRS to maintain accurate Leu- tRNALeu synthesis in the presence of norvaline, a non-canonical amino acid that accumulates in E. coli under microaerobic growth conditions. We found that LeuRS CP1 domain contains an efficient post-transfer editing site that hydrolyzes Nva- tRNALeu. Double-label experiments did not detect tRNA-dependent pre-transfer editing, and thus confirmed that norvaline is eliminated via proficient post-transfer pathway. The CP1 editing site also performs Ile-tRNALeu hydrolysis in a deacylation assay. However, to our surprise, we find that LeuRS discriminates against ultra-pure isoleucine at the activation step, with a discrimination factor as high as 31000, thereby obviating the need for post-transfer editing of isoleucine. We suggest that the prevailing opinion that LeuRS frequently misactivates isoleucine is an artifact of impure isoleucine samples. Using complementary kinetic, thermodynamic and structural approaches, we show that isoleucine is discriminated at both ground state binding and reaction kinetics. The result is also confirmed in vivo. Finally, we conclude that LeuRS editing operates to exclude norvaline from protein synthesis, and thus preserves the canonical genetic code under abnormal growth conditions.