Abstract
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 Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km 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 (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 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.
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Abbreviations
- ADP :
-
Adenosine diphosphate
- ATP :
-
Adenosine triphosphate
- BSA :
-
Bovine serum albumin
- CK :
-
Choline kinase
- CDP-Choline :
-
Cytidine 5’-diphosphocholine
- CDP-Etn :
-
Cytidine 5’-diphosphoethanolamine
- EDTA :
-
Ethylenediaminetetraacetic acid
- EhCK :
-
E. histolytica Choline kinase
- EhEK :
-
E. histolytica Ethanolamine kinase
- EK :
-
Ethanolamine kinase
- GST :
-
Glutathione-S-transferase
- hCK :
-
Human choline kinase
- hEK :
-
Human ethanolamine kinase
- His- :
-
6X histidine tag
- IPTG :
-
Isopropyl β-D-1-thiogalactopyranoside
- LB :
-
Luria-Bertani (medium)
- MBP :
-
Maltose binding protein
- NADH :
-
Reduced nicotinamide adenine dinucleotide
- ORF :
-
Open reading frame
- PCho :
-
Phosphocholine
- PCR :
-
Polymerase chain reaction
- PEtn :
-
Phosphoethanolamine
- PSD :
-
Phosphatidylserine decarboxylase
- PtdCho :
-
Phosphatidylcholine
- PtdEtn :
-
Phosphatidylethanolamine
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Acknowledgements
We would like to thank the laboratory staff of the School of Health Sciences for their technical assistance.
Funding
This work was supported by the Malaysia Ministry of Higher Education Fundamental Research Grant Scheme (FRGS), FRGS/1/2021/SKK0/USM/02/34 (203/PPSK/6171299).
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Ling Ling Few, Boon Huat Lim, Get Bee Yvonne-Tee, Ai Lan Chew, and Wei Cun See Too contributed to the study conception and design. Material preparation, data collection, and analyses were performed by Chiat Han Chang and Wei Cun See Too. The first draft of the manuscript was written by Chiat Han Chang and Ling Ling Few. All authors read and approved the final manuscript.
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Chang, C.H., Few, L.L., Lim, B.H. et al. Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms. Parasitol Res 122, 1651–1661 (2023). https://doi.org/10.1007/s00436-023-07869-5
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DOI: https://doi.org/10.1007/s00436-023-07869-5