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 Mn²⁺ over the typical Mg²⁺ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn²⁺, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg²⁺. Specifically, in Mg²⁺, EhCK1 exhibited a V_max and K_0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn²⁺, it displayed a V_max of 149.1 ± 2.5 U/mg and a K_0.5 of 9.5 ± 0.1 mM. Moreover, when Mg²⁺ was present at a constant concentration of 12 mM, the K_0.5 value for Mn²⁺ was ~ 2.4-fold lower than that in Mn²⁺ alone, without affecting its V_max. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn²⁺, it is worth noting that its K_m for choline and ATP were higher than in equimolar of Mg²⁺ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn²⁺, exhibiting Michaelis-Menten kinetic with ethanolamine (K_m = 312 ± 27 µM) and cooperativity with ATP (K_0.5 = 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 Mg²⁺, while choline kinase β differentially recognized choline and ethanolamine in Mg²⁺ and Mn²⁺, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn²⁺ 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.