Structural and kinetic profiling of Leishmania braziliensis trypanothione reductase: A molecular model for the development of targeted therapies.

This study explores the recombinant protein expression, purification, and characterization of Leishmania braziliensis Trypanothione Reductase (LbTR), an essential enzyme implicated in cutaneous leishmaniasis. Using E. coli as the host organism, the synthetic gene encoding LbTR was successfully expressed and subsequently purified using Immobilized Metal Affinity Chromatography (IMAC) yielding 20 mg/L of highly pure LbTR, verified by SDS-PAGE and isoelectric focusing. Comprehensive biochemical analyses were conducted to determine the recombinant enzyme's kinetic properties and structural features. Enzymatic assays revealed that LbTR efficiently reduces its natural substrate, following Michaelis-Menten kinetics. Structural characterization, including dynamic light scattering and fluorescence spectroscopy, confirmed the protein's stability and homogeneity in solution under varying temperatures. Circular dichroism analysis corroborated the presence of significant α-helical and β-sheet content, aligning with the structural model generated with AlphaFold. Molecular dynamics simulations over 1 μs were employed to investigate the conformational dynamics of LbTR in its native homodimeric form, complexed with essential cofactors and substrates. The results from these simulations offer valuable insights into the enzyme's structural behavior and catalytic mechanism, underscoring its potential as a target for therapeutic development against leishmaniasis.