Insights into the enzyme kinetics, structural features, and in silico inhibition of Leishmania donovani prolidase.

Abstract

Prolidase is a specialized metalloprotease that overcomes the resistance of proline-containing peptide bonds to proteolysis by exclusively hydrolyzing dipeptides with a C-terminal proline. Despite its potential significance in the life cycle of protozoan parasites, knowledge of prolidase in these organisms remains limited. In this study, the complete coding sequence of Leishmania donovani prolidase (LdProl) was cloned into an expression vector. The recombinant protein was purified using affinity chromatography, and its molecular weight was assessed by size-exclusion chromatography. The purified LdProl exhibited enzymatic activity with a strong substrate preference for Trp-Pro-pNA, and manganese ions were identified as essential cofactors for optimal activity. LdProl also stimulated innate immune mechanisms through elevating pro-inflammatory cytokines and nitric oxide levels, suggesting a role in macrophage-mediated immunomodulation. Fluorescence spectroscopy revealed pH-dependent conformational changes, particularly under strongly acidic conditions, whereas circular dichroism spectroscopy indicated a predominantly α-helical structure that was most stable at neutral pH. Computational modeling demonstrated a conserved three-dimensional structure, metal-binding site, and stable dimeric interface similar to prolidases from other organisms. Docking studies identified Trp-Pro-pNA as the substrate with the highest binding affinity, and molecular dynamics simulations confirmed the formation of stable enzyme-substrate complexes. Virtual screening yielded two lead compounds with higher binding affinities for LdProl than for its human counterpart, along with favorable ADME properties. Molecular dynamics simulations further validated these compounds by demonstrating the stability of enzyme-inhibitor complexes. Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.