Distal Domains of the Bacterial-Exclusive Wobble-Modifying Enzyme TilS Contribute to Catalysis

tRNA^Ile2 lysidine synthetase (TilS) is a bacterial-specific wobble-modifying enzyme that acts on the isoleucine-accepting tRNA^Ile2 _CAU. TilS installs a lysine at the C34 position of the anticodon, generating the lysidine modification. The resulting LAU anticodon enables exclusive decoding of infrequently used AUA isoleucine codons, rejecting AUG methionine codons. Compared to other wobble-modifying enzymes that contact only the anticodon arm of their cognate tRNAs, TilS is distinct in containing additional domains outside of the N-terminal active site. For type I TilS enzymes such as the B. cenocepacia TilS (BcTilS) investigated here, appended domains contact the tRNA^Ile2 substrate along the body and through the acceptor stem, up to 60 Å away from the target C34. Among bacterial tRNAs, only unmodified tRNA^Ile2 and tRNA^Met share an anticodon, suggesting that the appended domains of TilS provide substrate recognition strategies that other wobble-modifying enzymes do not need. Here, we investigate both protein and tRNA elements to understand the strategy by which TilS accepts its cognate tRNA^Ile2 substrate and rejects the near-cognate tRNA^Met.