The
flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an
oxidoreductase that uses
NAD(P)H as cofactor. This
enzyme is clinically relevant due to its role in the action mechanism of some
trypanocidal drugs used in the treatment of
Chagas' disease by producing
reactive oxygen species. In this work, the recombinant
enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/β)8 fold with the
FMN prosthetic group buried at the positively-charged active-site cleft. In
solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic
protein flexibility. Moreover, the binding mode of β-
lapachone, a trypanocidal agent, and other
naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the
isoalloxazine ring from
FMN and residues from the active-site pocket.