title: An alternative mechanism for the methylation of phosphoethanolamine catalyzed by Plasmodium falciparum phosphoethanolamine methyltransferase.
abstract: The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19-His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical Sn2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.
authors.affiliation: From the Joint Barcelona Supercomputing Center-Centre for Genomic Regulation-Institute for Research in Biomedicine Research Program, Carrer de Jordi Girona 29, 08034 Barcelona, Spain.
authors.forename: Suwipa
authors.initials: S
authors.lastname: Saen-Oon
authors.affiliation: the Department of Biology, Washington University, St. Louis, Missouri 63130, and.
authors.forename: Soon Goo
authors.initials: SG
authors.lastname: Lee
authors.affiliation: the Department of Biology, Washington University, St. Louis, Missouri 63130, and jjez@biology2.wustl.edu.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
authors.affiliation: From the Joint Barcelona Supercomputing Center-Centre for Genomic Regulation-Institute for Research in Biomedicine Research Program, Carrer de Jordi Girona 29, 08034 Barcelona, Spain, the Catalan Institution for Research and Advanced Studies, Barcelona 08010, Spain victor.guallar@bsc.es.
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry* / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010802:Phylogeny; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000201:enzymology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D004983:Ethanolamines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; C005448:phosphorylethanolamine; D008780:Methyltransferases; C102844:phosphoethanolamine methyltransferase
keywords: Computer Modeling; Crystallography; Enzyme Mechanism; Mutagenesis; Protein Structure