Molecular Analysis of Site‐directed Mutants of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum

Abstract

Plasmodium falciparum, which is responsible for malaria in humans, requires rapid division and efficient phospholipid synthesis to survive. Unlike mammals, Plasmodium require phosphoethanolamine methyltransferases (PfPMT) to synthesize phosphocholine for membrane biogenesis. Disruption of the PfPMT gene causes severe growth, reproduction, and viability defects. Thus, this enzyme is a potential anti‐malarial target. To examine the role of residues in the phosphobase binding site and a potentially catalytic histidine in PfPMT, site‐directed mutagenesis, functional assays, and protein crystallography were used. Point mutants targeting the phosphobase binding site and the catalytic histidine were expressed in E. coli and purified by affinity chromatography. Assays of the phosphobase site mutants indicate that electrostatic interactions provided by Arg179 and Lys247 are required for activity. Tyr27, Tyr160, Tyr175, and Tyr181 are also functionally important. Mutation of His132 resulted in proteins with no detectable activity; however, these proteins still bound substrates with affinity comparable to wild‐type. To further investigate the structure of this mutant, the H132A protein was crystallized and a 1.19 Å resolution data set collected. The resulting structure supports a critical role for the histidine in the reaction mechanism. Research funded by the Howard Hughes Medical Institute.