Malaria is a debilitating parasitic disease. Each year 200 million new infections of Plasmodium falciparum malaria are established, resulting in 0.4 million deaths worldwide. Resistance to the frontline artemisinin combination therapy (ACT) is apparent in South East Asia, requiring the urgent development of new therapies with novel mechanisms of action. Protein translation is essential for the survival of P. falciparum and offers a promising new avenue for drug development.
The aim of the current work is to characterise P. falciparum Tyrosyl-tRNA Synthetase (PfYRS) as a target for new inhibitors and identify key differences from the human counterpart (HsYRS). The crystal structure of recombinant PfYRS bound to the tyrosyl-adenylate intermediate (Tyr-AMP) was solved at 1.8 Å resolution. We established a luciferase-based assay to measure ATP consumption in the first step of the PfYRS reaction. Using differential scanning fluorimetry (DSF), we showed that the melting temperature (Tm) of PfYRS increases upon addition of ATP and tyrosine, consistent with the formation and tight binding of Tyr-AMP. We generated PftRNATyr and established an aminoacylation assay to measure formation of the final enzymatic product, Tyr-tRNATyr. Recombinant HsYRS was found to consume ATP at a faster rate to PfYRS, suggesting more efficient formation of the Tyr-AMP intermediate. Interestingly, the melting temperature of HsYRS was not increased upon addition of ATP and tyrosine, suggesting that Tyr-AMP does not bind as tightly to the human enzyme. Analysis of the protein structure revealed that the KMSKS loop, important for access to the active site, was well resolved in the structure of PfYRS but is flexible in the published HsYRS structure (4QBT). The more highly structured KMSSS loop in PfYRS may be contributing to the tighter binding of Tyr-AMP, and this difference can be exploited for the development of new antimalarial drugs.