Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.

Streptococcus pneumoniae causes bacterial pneumonia with high mortality and morbidity. The emergency of multidrug-resistant bacteria threatens the treatment of the disease. Leucyl-tRNA synthetase (LeuRS) plays an essential role in cellular translation and is an attractive drug target for antimicrobial development. Here we report the compound ZCL039, a benzoxaborole-based derivative of AN2690, as a potent anti-pneumococcal agent that inhibits S. pneumoniae LeuRS (SpLeuRS) activity. We show using kinetic, biochemical analyses combined with the crystal structure of ZCL039-AMP in complex with the separated SpLeuRS editing domain, that ZCL039 binds to the LeuRS editing active site which requires the presence of tRNALeu, and employs an uncompetitive inhibition mechanism. Further docking models establish that ZCL039 clashes with the eukaryal/archaeal specific insertion I4ae helix within editing domains. These findings demonstrate the potential of benzoxaboroles as effective LeuRS inhibitors for pneumococcus infection therapy, and provide future structure-guided drug design and optimization.