1. Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases
- Author
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Terry K. Smith, Federica Gibellini, William N. Hunter, BMS, University of St Andrews [Scotland], The Wellcome Trust, University of St Andrews. School of Biology, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
Choline kinase ,GPCho, glycerophosphocholine ,QH301 Biology ,Drug target ,Expression ,Saccharomyces-cerevisiae ,Tb, Trypanosome brucei ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Ethanolamine Kinase 1 ,GPSer, glycerophosphoserine ,Choline Kinase ,Caenorhabditis-elegans ,Choline ,QD ,Trypanosoma brucei ,HPTLC, high-performance TLC ,Cloning, Molecular ,GPEtn, glycerophosphoethanolamine ,Purification ,Phosphocholine ,African sleeping sickness ,0303 health sciences ,RT, reverse transcription ,biology ,Ethanolamine kinase ,030302 biochemistry & molecular biology ,Life Sciences ,MALDI, matrix-assisted laser-desorption ionization ,VSG, variant-surface glycoprotein ,C/EK, choline/ethanolamine kinase ,UTR, untranslated region ,Phosphotransferases (Alcohol Group Acceptor) ,LB, Luria–Bertani ,Kennedy pathway ,Specificity ,Donor ,Research Article ,GPI, glycosylphosphatidylinositol ,Molecular Sequence Data ,Trypanosoma brucei brucei ,TOF, time-of-flight ,TEV, tobacco etch virus ,ethanolamine kinase ,QH301 ,03 medical and health sciences ,Ethanolamine ,ORF, open reading frame ,SDG 3 - Good Health and Well-being ,PtdCho, phosphotidylcholine ,Animals ,Amino Acid Sequence ,Molecular Biology ,EK, ethanolamine kinase ,030304 developmental biology ,Phosphatidylethanolamine ,PtdEtn, phosphatidylethanolamine ,Cell Biology ,QD Chemistry ,biology.organism_classification ,glycosylphosphatidylinositol ,Kinetics ,chemistry ,Sequence Alignment - Abstract
Note related output below contains correction of this paper. Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (glycero-phosphoethanolamine) and GPCho (glycerophosphocholine). Ethanolamine is also found as an integral component of the GPI (glycosylpliosphatidylinositol) 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. TbEK 1 (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 K values for ethanolamine and ATP were found to be 18.4 +/- 0.9 and 219 29 mu M 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 K-m 80 times lower than that of ethanolamine. The K. values for choline, ethanolamine and ATP were 31.4 +/- 2.6 mu M, 2.56 +/- 0.31 mu M and 20.6 +/- 1.96 mu M 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 oil C-2, but substitutions oil C-1 and elongations of the carbon chain were not well tolerated. Publisher PDF
- Published
- 2008
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