Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms.

The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn over the typical Mg as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg. Specifically, in Mg, EhCK1 exhibited a V and K of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn, it displayed a V of 149.1 ± 2.5 U/mg and a K of 9.5 ± 0.1 mM. Moreover, when Mg was present at a constant concentration of 12 mM, the K value for Mn was ~ 2.4-fold lower than that in Mn alone, without affecting its V. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn, it is worth noting that its K for choline and ATP were higher than in equimolar of Mg in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn, exhibiting Michaelis-Menten kinetic with ethanolamine (K = 312 ± 27 µM) and cooperativity with ATP (K = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg, while choline kinase β differentially recognized choline and ethanolamine in Mg and Mn, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.