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1.  Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases 
Biochemical Journal  2008;415(Pt 1):135-144.
Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycerophosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4±0.9 and 219±29 μM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.
PMCID: PMC2552378  PMID: 18489261
choline kinase; ethanolamine kinase; Kennedy pathway; Trypanosoma brucei; C/EK, choline/ethanolamine kinase; EK, ethanolamine kinase; GPCho, glycerophosphocholine; GPEtn, glycerophosphoethanolamine; GPI, glycosylphosphatidylinositol; GPSer, glycerophosphoserine; HPTLC, high-performance TLC; LB, Luria–Bertani; MALDI, matrix-assisted laser-desorption ionization; ORF, open reading frame; PtdCho, phosphotidylcholine; PtdEtn, phosphatidylethanolamine; RT, reverse transcription; Tb, Trypanosome brucei; TEV, tobacco etch virus; TOF, time-of-flight; UTR, untranslated region; VSG, variant-surface glycoprotein
2.  Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate 
Molecular Microbiology  2006;61(6):1457-1468.
The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
PMCID: PMC1618733  PMID: 16968221
3.  The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei 
Molecular Microbiology  2009;73(5):826-843.
Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine-phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI-MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.
PMCID: PMC2784872  PMID: 19555461

Results 1-3 (3)