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Title

The Phosphobase Methylation Pathway in Plants, Nematodes, and Protozoan Plasmodium falciparum: Phosphoethanolamine N-methyltransferase

Date of Award

Summer 8-15-2012

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Phosphatidylcholine (PtdCho) is a major phospholipid component in the cellular membranes of eukaryotes. Depending on the organism, the biosynthesis of PtdCho occurs through three metabolic routes, which are Bremer-Greenberg pathway, Kennedy pathway, and phosphobase methylation pathway. In plants, nematodes and protozoan parasite Plasmodium, the phosphobase methylation pathway is the major route for PtdCho biosynthesis. In the phosphobase methylation pathway, phosphoethanolamine N-methyltransferase (PMT) catalyzes the triple sequential methylation of phosphoethanolamine (pEA) to produce phosphocholine (pCho), a precursor of PtdCho. Unlike the biosynthetic pathways of mammals and yeast, the PMT-mediated phosphobase methylation biosynthesis is not well studied.

My thesis work examines the PMT enzymes and pathways of PtdCho biosynthesis in Arabidopsis thaliana (type I plant PMT), Plasmodium falciparum (type II Plasmodium PMT), and Haemonchus contortus (type III nematode PMT) by a combination of structural biology and biochemical approaches.

The first part of my thesis examines the biochemical funtion of recently identified type III nematode PMT. To determine if the PMT from a parasitic nematode share the same domain organization and biochemical reaction specificity as the previously described enzymes from Caenorhabditis elegans, two PMT homologues (HcPMT1 and HcPMT2) were cloned from Haemonchus contortus (sheep barber pole worm). Biochemical studies using radiochemical enzymatic assays and isothermal titration calorimetry (ITC) experiments of the two HcPMT revealed key similarities and differences. Through the biochemical characterization, we found that the nematode PMT are well conserved and that the two specific catalytic domains may undergo different conformational changes upon ligand binding.

The second part of my thesis uses protein crystallography to understand the molecular details of the PMT. The three-dimensional structures of the type II Plasmodium PMT in complex with various ligands (substrates, product, and inhibitors) were determined by X-ray crystallography. Studies of a series of PfPMT structures and site-directed mutants indicate that a catalytic dyad, Tyr19 and His132, is critical for catalysis and substrate recognition. Based on the functional and crystallographic analyses, a reaction mechanism for PfPMT is proposed. As a follow-up study, the crystal structure of PfPMT in complex with a potential small molecule inhibitor, amodiaquine (AQ), was solved. With the crystallographic analysis of PfPMT*AQ, the IC50 values of several aminoquinolines and enzymatic activities of site-directed mutants were determined. These studies offer a possible approach for the development of PfPMT inhibitors.

In the final section of my thesis, a comprehensive structural comparison of the plant and nematode PMT was conducted. To understand the organization of active sites, reaction chemistry, and substrate specificity in each PMT enzyme, X-ray crystal structures of Arabidopsis thaliana PMT (type I plant PMT), HcPMT1 (type III nematode PMT), and HcPMT2 (type III nematode PMT) were determined. The crystallographic analysis of overall folds and active site structures reveals that different sets of catalytic residues catalyze the same reaction chemistry in the PMT1 and PMT2 domains. In addition, structural differences of the phosphobase binding pockets explain the highly selective substrate discrimination of PMT1 and PMT2.

Overall, my thesis work provides insight on the functional properties, the reaction mechanisms, and the active site structures of all types of PMT enzymes that are essential for survival of the organisms. Since the PMT homologs are not found in mammals and the PMT enzymes share less than 25% homology with other types of PMT, my thesis work allows us to develop small molecule inhibitors against parasite PMT enzymes. Collectively, my thesis studies on the PMT enzymes could be valuable in human and veterinary medicine and agriculture.

Language

English (en)

Chair and Committee

Joseph M. Jez

Committee Members

Jan G. Jaworski, Robert G. Kranz, Barbara N. Kunkel, Sona Pandey, Thomas J. Smith

Comments

Permanent URL: https://doi.org/10.7936/K7416V0F

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