Biochemical and genetic analysis of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum.
The PfPMT enzyme of Plasmodium falciparum, the agent of severe human malaria, is a member of a large family of known and predicted phosphoethanolamine methyltransferases (PMTs) recently identified in plants, worms, and protozoa. Functional studies in P. falciparum revealed that PfPMT plays a critical role in the synthesis of phosphatidylcholine via a plant-like pathway involving serine decarboxylation and phosphoethanolamine methylation. Despite their important biological functions, PMT structures have not yet been solved, and nothing is known about which amino acids in these enzymes are critical for catalysis and binding to S-adenosyl-methionine and phosphoethanolamine substrates. Here we have performed a mutational analysis of PfPMT focused on 24 residues within and outside the predicted catalytic motif. The ability of PfPMT to complement the choline auxotrophy of a yeast mutant defective in phospholipid methylation enabled us to characterize the activity of the PfPMT mutants. Mutations in residues Asp-61, Gly-83 and Asp-128 dramatically altered PfPMT activity and its complementation of the yeast mutant. Our analyses identify the importance of these residues in PfPMT activity and set the stage for advanced structural understanding of this class of enzymes.
Reynolds
Jennifer M
JM
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Framington, CT 06030, USA.
Takebe
Sachiko
S
Choi
Jae-Yeon
JY
El Bissati
Kamal
K
Witola
William H
WH
Bobenchik
April M
AM
Hoch
Jeffrey C
JC
Voelker
Dennis R
DR
Mamoun
Choukri Ben
CB
D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D004983:Ethanolamines / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D016778:Malaria, Falciparum / Q000201:enzymology / Q000235:genetics; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D010713:Phosphatidylcholines / Q000096:biosynthesis; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D004983:Ethanolamines; D010713:Phosphatidylcholines; D015800:Protozoan Proteins; C005448:phosphorylethanolamine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C102844:phosphoethanolamine methyltransferase