title: The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active.
abstract: Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 μM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.
authors.affiliation: Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G2W1.
authors.forename: Huogen
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G2W1.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Bryksa
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
authors.forename: Prasenjit
authors.initials: P
authors.lastname: Bhaumik
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.affiliation: Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
authors.affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive, Singapore 117543, Singapore.
authors.forename: Shao Q
authors.initials: SQ
authors.lastname: Yao
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.affiliation: Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON, Canada N1G2W1; Faculty of Land and Food Systems, University of British Columbia 248-2357 Main Mall Vancouver, BC V6T 1Z4. Electronic address: r.yada@ubc.ca.
authors.forename: Rickey Y
authors.initials: RY
authors.lastname: Yada
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D058849:Protein Refolding; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin
keywords: Plasmepsin; Plasmodium falciparum; Prosegment; Protease inhibitor; Refolding; Zymogen