Purification and functional analysis of the Mycobacterium leprae thioredoxin/thioredoxin reductase hybrid protein.

In Mycobacterium leprae, thioredoxin and thioredoxin reductase are expressed 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 thioredoxin reductase (TR) are expressed as two separate proteins. This raises the question of whether the hybrid protein is enzymatically active and, if so, whether TR reduces its own Trx partner or alternatively a heterologous Trx subunit. To address this question, the hybrid TR/Trx protein of M. leprae as well as the individual parts of the hybrid gene coding for either TR or Trx were overexpressed in Escherichia coli 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. Compared with the enzymatic activity of the separately expressed Trx and TR proteins, the hybrid protein is shown to be more efficient, particularly at low equimolar concentrations. This suggests that the hybrid protein of M. leprae is active by itself and that its activity involves intramolecular interactions between the TR and Trx domains. The activity of the hybrid protein increases when exogenous TR or Trx is added, indicating an additional role for intermolecular interactions.

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae.

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.