A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases.

The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA esterification and allows fidelity in protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from Plasmodium falciparum to determine the basis of their specificity towards L-asp and L-asn respectively. The negatively charged L-asp and its analogue L-asn differ only in their side-chain groups i.e., -OH and -NH2. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (L-asp/L-asn) binding sites across species revealed two highly conserved residues in PfDRS (D408 and K372) and PfNRS (E395 and L360) that are involved in recognition of the Oδ2/Nδ2 of L-asp/L-asn respectively. These residues were mutated and swapped between the D408→E in PfDRS and the corresponding E395→D in PfNRS. A similar approach was employed for residue number K372→L in PfDRS and L360→K in PfNRS. The mutated PfDRSD408E retained its enzymatic activity during step 1 of aminoacylation reaction towards L-asp and L-asn and esterified tRNAAsp with L-asp like wild type enzyme, while the PfDRSK372L was rendered enzymatically inactive. The correspondingly mutated PfNRSE395D was enzymatically inactive. The mutated PfNRSL360K had an altered specificity and esterified tRNAAsn with non-cognate amino acid L-asp and not L-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of PfDRS while the residue L360 in PfNRS imparts specificity towards L-asn.