Differences in Charge Distribution in Leishmanolysin Result in a Reduced Enzymatic Activity.

is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in genome, but no proteolytic activity was detected. Here, we analyzed leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, and leishmanolysins were cloned in a pLEXSY expression system into . Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that leishmanolysins harbor a weak enzymatic activity about three times less abundant than leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.