Plasmodium falciparum Plasmepsin V (PfPMV): Insights into recombinant expression, substrate specificity and active site structure.

Abstract Plasmepsin V from Plasmodium falciparum ( Pf PMV) is responsible for the cleavage of the Plasmodium export element (PEXEL) motif at the N-terminus of several hundreds of the exported proteins. Pf PMV is necessary for parasite viability and has become a novel promising target for antimalarial therapy. The first recombinant expression of soluble, active Pf PMV as thioredoxin fusion proteins is reported herein. Two truncated forms of Pf PMV were fused to thioredoxin (Trx) to generate Trx- Pf PMVp37 and Trx- Pf PMVm84. The fusion proteins were successfully purified using Ni 2+ affinity chromatography in combination with ATP treatment to eliminate Escherichia coli HSP60 contaminant. Trx- Pf PMVm84 could hydrolyze the PEXEL-containing peptides more efficiently than Trx- Pf PMVp37. Interestingly, both Trx- Pf PMVs preferred to cleave Pf EMP2 peptide over HRPII peptide. The replacement of Ser with Val or Glu at P 1 ′ position created a substrate with 75% reduction in the enzyme activity, whereas the substitution of Ile with Lys or Glu at P 2 position reduced the cleavage efficiency by 30%. The activity of Trx- Pf PMVm84 was inhibited by PMSF and nelfinavir but not by pepstatin A. After the removal of Trx domain, activities of both enzymes toward Pf EMP2 and HRPII peptides were fitted to the Michaelis-Menten model to determine kinetic parameters. The K m values toward both peptides were apparently much lower than the previously reported data although with similar k cat values. Along with an improved Pf PMV preparation protocol, these findings have provided insights into its substrate specificity at P 2 and P 1 ′ positions as well as interactions among the enzyme, substrates, and inhibitors.