Abstract
The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.
Highlights
Coenzyme A (CoA) is an essential cofactor in all living organisms as an acyl group carrier and carbonyl-activating group involved in more than 100 cellular reactions (Begley et al, 2001); it is estimated to be a cofactor used in 9% of identified enzymatic reactions (Strauss, 2010)
We identified all putative genes involved in CoA biosynthesis in the reference genome of E. histolytica: HM-1:IMSS (Fig. 1)
We identified four enzymes that are involved in CoA synthesis in E. histolytica (Fig. 1) and characterized the first rate-limiting enzyme in this process, PanK
Summary
Coenzyme A (CoA) is an essential cofactor in all living organisms as an acyl group carrier and carbonyl-activating group involved in more than 100 cellular reactions (Begley et al, 2001); it is estimated to be a cofactor used in 9% of identified enzymatic reactions (Strauss, 2010). CoA is synthesized from pantothenate (vitamin B5), cysteine, and ATP (Jackowski, 1996; Leonardi et al, 2005b). Entamoeba histolytica is the protozoan agent responsible for human amebiasis, an infectious disease causing dysentery and amebic liver abscesses, responsible for 100,000 deaths annually throughout the world. It represents the third most common parasitic cause of death, after malaria and schistosomiasis (Stanley, 2003; Ali and Nozaki, 2007; Ralston and Petri, 2011). The molecular target of metronidazole has been well described as a key metabolic enzyme, pyruvate: ferredoxin oxidoreductase, which is involved in acetyl CoA production from pyruvate as part of central energy metabolism. Identification and characterization of novel drug targets unique to E. histolytica are needed to design better therapeutics against amebiasis
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