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J. Biol. Chem., Vol. 283, Issue 12, 7894-7900, March 21, 2008

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Biochemical and Genetic Analysis of the Phosphoethanolamine Methyltransferase of the Human Malaria Parasite Plasmodium falciparum^*

Jennifer M. Reynolds¹, Sachiko Takebe¹, Jae-Yeon Choi, Kamal El Bissati, William H. Witola, April M. Bobenchik, Jeffrey C. Hoch^¶, Dennis R. Voelker, and Choukri Ben Mamoun²

From the Department of Genetics and Developmental Biology and the ^¶Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030 and the Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206

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.

Received for publication, December 4, 2007 , and in revised form, January 2, 2008.

^* This research was supported by National Institutes of Health and Department of Defense Grants AI51507, AI58962, and PR033005 and Burroughs Wellcome Fund Awards 1006267 (to C. B. M.) and 5R37GM32453 (to D. R. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Recipient of the Burroughs Wellcome Award, Investigators of Pathogenesis of Infectious Disease. To whom correspondence should be addressed: Dept. of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-3301. Tel.: 860-679-3544; Fax: 860-679-8345; E-mail: Choukri{at}up.uchc.edu.

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