The rodent
malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human
malaria. Plasmepsins are a class of
aspartic proteinase isoforms that exert multiple pathological effects in
malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze
hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV
plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature
enzyme from genomic
DNA. We over-expressed this PbPM4
zymogen as inclusion bodies (IB) in Escherichia coli, and purified the
protein following in vitro IB refolding. Auto-maturation of the PbPM4
zymogen to mature
enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4
zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied
enzyme kinetics on substrates and inhibitors of
aspartic proteinases. Using combinatorial chemistry-based
peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective
peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human
malaria parasites, and human
cathepsin D (hcatD). We showed that this compound exhibited
a >10-fold selectivity to PbPM4 and human
malaria parasite
plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the
plasmepsin family and provides leads for
anti-malarial drug design.