Abstract

The path to survival for pathogenic organisms is not straightforward. Pathogens require a set of enzymes for tissue damage generation and to obtain nourishment, as well as a toolbox full of alternatives to bypass host defense mechanisms. Our group has shown that the parasitic protist Entamoeba histolytica encodes for 14 sphingomyelinases (SMases); one of them (acid sphingomyelinase 6, aSMase6) is involved in repairing membrane damage and exhibits hemolytic activity. The enzymatic characterization of aSMase6 has been shown to be activated by magnesium ions but not by zinc, as shown for the human aSMase, and is strongly inhibited by cobalt. However, no structural data are available for the aSMase6 enzyme. In this work, bioinformatic analyses showed that the protist aSMases are diverse enzymes, are evolutionarily related to hemolysins derived from bacteria, and showed a similar overall structure as parasitic, free-living protists and mammalian enzymes. AlphaFold3 models predicted the occupancy of cobalt ions in the active site of the aSMase6 enzyme. Cavity blind docking showed that the substrate is pushed outward of the active site when cobalt is bound instead of magnesium ions. Additionally, the structural models of the aSMase6 of E. histolytica showed a loop that is absent from the rest of the aSMases, suggesting that it may be involved in hemolytic activity, as demonstrated experimentally using the recombinant proteins of aSMase4 and aSMase6. Trichomonas vaginalis enzymes show a putative transmembrane domain and seem functionally different from E. histolytica. This work provides insight into the future biochemical analyses that can show mechanistic features of parasitic protists sphingomyelinases, ultimately rendering these enzymes potential therapeutic targets.