PURIFICATION AND FUNCTIONAL-ANALYSIS OF THE MYCOBACTERIUM-LEPRAE THIOREDOXIN THIOREDOXIN REDUCTASE HYBRID PROTEIN

In Mycobacterium leprae, thioredoxin and thioredoxin reductase are exp ressed from a single gene. This results in the expression of a hybrid protein with subunits attached to each other by a hydrophilic peptide linker. In all other organisms studied so far, thioredoxin (Trx) and t hioredoxin reductase (TR) are expressed as two separate proteins. This raises the question of whether the hybrid protein is enzymatically ac tive and, if so, whether TR reduces its own Trx partner or alternative ly a heterologous Trx subunit. To address this question, the hybrid TR /Trx protein of M. leprae as well as the individual parts of the hybri d gene coding for either TR or Trx were overexpressed in Escherichia c oli and purified. The purified proteins were tested for their ability to catalyze NADPH-dependent insulin disulfide reduction. Here we show that the M. leprae hybrid protein is indeed enzymatically active. Comp ared with the enzymatic activity of the separately expressed Trx and T R proteins, the hybrid protein is shown to be more efficient, particul arly at low equimolar concentrations. This suggests that the hybrid pr otein of M. leprae is active by itself and that its activity involves intramolecular interactions between the TR and Trx domains. The activi ty of the hybrid protein increases when exogenous TR or Trx is added, indicating an additional role for intermolecular interactions.