Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases

Abstract

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 K m 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 K m 80 times lower than that of ethanolamine. The K m 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.

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library Against Trypanosoma brucei Choline Kinase; A Genetically Validated Drug Target

“…al. [39], using TbCK and radiolabelled choline has been performed previously [23]. However this is not suitable for screening purposes, so choline kinase activity was measured by a spectrophotometric coupled assay (Figure 1).…”

“…Large-scale recombinant expression and purification of TbCK was conducted using the construct pET-15bTEV-TbCK in BL21 Rosetta (DE3) cells as described previously [23], except the cells were grown in tryptone phosphate broth [37], harvested by centrifugation at 3500 g for 20 min at 4°C and affinity purified with either a HisTrap™ FF crude column (enzyme activity assay) or a HisTALON Cartridge (thermal shift analysis).…”

“…High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 μl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only.…”

“…This involves phosphorylation of ethanolamine by an ethanolamine kinase, its activation to CDP-ethanolamine by an ethanolamine-phosphate cytidyltransferase and its transfer to diacylglycerol by an ethanolamine phosphotransferase. The presence of this branch of the Kennedy pathway was demonstrated in T. brucei [22], however only recently have the constituent enzymes been characterized [23].…”

“…This involves the phosphorylation of choline by a choline kinase, its activation to CDP-choline by a choline-phosphate cytidyltransferase and its transfer to diacylglycerol by a choline phosphotransferase. Biochemical characterisation of the two choline/ethanolamine kinases involved in the initial steps of the Kennedy pathway show that unusually amongst eukaryotes only one of the kinases is able to phosphorylate choline [23].…”