Divergent Anticodon Recognition in Contrasting Glutamyl-tRNA Synthetases

The pathogenic bacterium Helicobacter pylori utilizes two essential glutamyl-tRNA synthetases (GluRS1 and GluRS2). These two enzymes are closely related in evolution and yet they aminoacylate contrasting tRNAs. GluRS1 is a canonical discriminating GluRS (D-GluRS) that biosynthesizes Glu-tRNAGlu and cannot make Glu-tRNAGln. In contrast, GluRS2 is non-canonical as it is only essential for the production of misacylated Glu-tRNAGln. The co-existence and evident divergence of these two enzymes was capitalized upon to directly examine how GluRS2 acquired tRNAGln specificity. One key feature that distinguishes tRNAGlu from tRNAGln is the third position in the anticodon of each tRNA (C36 versus G36, respectively). By comparing sequence alignments of different GluRSs, including GluRS1s and GluRS2s, to the crystal structure of the Thermus thermophilus D-GluRS:tRNAGlu complex, a divergent pattern of conservation in enzymes that aminoacylate tRNAGlu versus those specific for tRNAGln emerged and was experimentally validated. In particular, when an arginine conserved in discriminating GluRSs and GluRS1s was inserted into Hp GluRS2 (Glu334Arg GluRS2), the catalytic efficiency of the mutant enzyme (kcat/KMapp) was reduced by approximately one order of magnitude towards tRNAGln. However, this mutation did not introduce activity towards tRNAGlu. In contrast, disruption of a glycine that is conserved in all GluRS2s but not in other GluRSs (Gly417Thr GluRS2) generated a mutant GluRS2 with weak activity towards tRNAGlu1. Synergy between these two mutations was observed in the double mutant (Glu334Arg/Gly417Thr GluRS2), which specifically and more robustly aminoacylates tRNAGlu1 instead of tRNAGln. As GluRS1 and GluRS2 are related by an apparent gene duplication event, these results demonstrate that we can experimentally map critical evolutionary events in the emergence of new tRNA specificities.