title: Plasmodium falciparum Plasmepsin V (PfPMV): Insights into recombinant expression, substrate specificity and active site structure.
abstract: Plasmepsin V from Plasmodium falciparum (PfPMV) is responsible for the cleavage of the Plasmodium export element (PEXEL) motif at the N-terminus of several hundreds of the exported proteins. PfPMV is necessary for parasite viability and has become a novel promising target for antimalarial therapy. The first recombinant expression of soluble, active PfPMV as thioredoxin fusion proteins is reported herein. Two truncated forms of PfPMV were fused to thioredoxin (Trx) to generate Trx-PfPMVp37 and Trx-PfPMVm84. The fusion proteins were successfully purified using Ni(2+) affinity chromatography in combination with ATP treatment to eliminate Escherichia coli HSP60 contaminant. Trx-PfPMVm84 could hydrolyze the PEXEL-containing peptides more efficiently than Trx-PfPMVp37. Interestingly, both Trx-PfPMVs preferred to cleave PfEMP2 peptide over HRPII peptide. The replacement of Ser with Val or Glu at P1' position created a substrate with 75% reduction in the enzyme activity, whereas the substitution of Ile with Lys or Glu at P2 position reduced the cleavage efficiency by 30%. The activity of Trx-PfPMVm84 was inhibited by PMSF and nelfinavir but not by pepstatin A. After the removal of Trx domain, activities of both enzymes toward PfEMP2 and HRPII peptides were fitted to the Michaelis-Menten model to determine kinetic parameters. The Km values toward both peptides were apparently much lower than the previously reported data although with similar kcat values. Along with an improved PfPMV preparation protocol, these findings have provided insights into its substrate specificity at P2 and P1' positions as well as interactions among the enzyme, substrates, and inhibitors.
authors.affiliation: Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan road, Lat Yao, Chatuchak, Bangkok 10900, Thailand; Center of Excellence for Innovation in Chemistry, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan road, Lat Yao, Chatuchak, Bangkok 10900, Thailand. Electronic address: nonlawat.b@ku.ac.th.
authors.forename: Nonlawat
authors.initials: N
authors.lastname: Boonyalai
authors.affiliation: Department of Biochemistry and Special Research Unit for Protein Engineering and Protein Bioinformatics, Faculty of Science, Kasetsart University, 50 Ngam Wong Wan road, Lat Yao, Chatuchak, Bangkok 10900, Thailand.
authors.forename: Pichamon
authors.initials: P
authors.lastname: Sittikul
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 road, Phayathai, Bangkok 10400, Thailand.
authors.forename: Jirundon
authors.initials: J
authors.lastname: Yuvaniyama
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin
keywords: Export; Malaria; PEXEL; Plasmepsin V