Functional characterization of PfHDDCP reveals a HotDog-domain acyl‑CoA thioesterase as a potential antimalarial drug target

Thioesterases play a central role in maintaining acyl‑CoA homeostasis and lipid metabolic flux, processes that are critical for the intraerythrocytic development of Plasmodium falciparum. Plasmodium falciparum HotDog domain-containing protein [PfHDDCP (PF3D7_1135400; previously annotated as PF11_0364, XP_001348035.1)] has been computationally predicted to function as an acyl-CoA thioesterase, although experimental validation was lacking. Here, we present the first biochemical characterization of PfHDDCP and demonstrate its thioesterase activity.

The PfHDDCP gene was cloned into the pRSET-A vector, expressed in Escherichia coli BL21(DE3), purified under denaturing conditions, and refolded. Enzymatic assays revealed no detectable protease activity but demonstrated robust thioesterase activity toward acetyl-CoA using a DTNB-based colorimetric assay. Kinetic analysis yielded a Km of 11.68 µM and a Vmax of 16.28 × 10⁻⁵ µmol/min, with substrate inhibition at elevated acetyl-CoA concentrations. PfHDDCP showed maximal catalytic activity at pH 8.0 and 37 °C and retained substantial activity at 40-42 °C. Fe²⁺ and Ca²⁺ had no significant effect on activity, whereas Mg²⁺ markedly reduced activity at the higher concentrations tested. Molecular docking predicted favorable heme binding, but neither spectroscopy nor native PAGE supported stable heme association in vitro. Artemisinin derivatives showed favorable predicted interactions with PfHDDCP and inhibited its thioesterase activity in vitro, whereas chloroquine had a substantially weaker effect.

Collectively, these findings establish PfHDDCP as a functional HotDog domain-containing acyl-CoA thioesterase and support its potential involvement in parasite lipid metabolism. The inhibition observed with selected antimalarial compounds identifies PfHDDCP as a candidate for further investigation of drug-enzyme interactions.

Not Applicable.