Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3′-Kinase

• Published in: Biochemistry (Easton) Vol. 56; no. 33; pp. 4335 - 4345
• Main Authors: Hassett, Matthew R, Sternberg, Anna R, Riegel, Bryce E, Thomas, Craig J, Roepe, Paul D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22-08-2017
• Subjects: Class III Phosphatidylinositol 3-Kinases - biosynthesis; Class III Phosphatidylinositol 3-Kinases - chemistry; Class III Phosphatidylinositol 3-Kinases - genetics; Class III Phosphatidylinositol 3-Kinases - isolation & purification; Cloning, Molecular; Gene Expression; Plasmodium falciparum - enzymology; Plasmodium falciparum - genetics; Protein Domains; Protozoan Proteins - biosynthesis; Protozoan Proteins - chemistry; Protozoan Proteins - isolation & purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.7b00416

The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3′-kinase (PI3K), and sequence analysis suggests that the enzyme is a “class III”- or “Vps34”-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3′-phosphate production from phosphatidylinositol (Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme’s catalytic and “N-lobe” domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs’ endoperoxide bridge to generate reactive alkylating agents.