(Received for publication, July 17, 1995; and in revised form, August 21, 1995)
From the
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.
Mycobacterium leprae, the causative agent of leprosy,
is an intracellular pathogen that cannot be cultured in vitro.
By screening of recombinant DNA libraries (1, 2) and
subsequent gene analysis, a large number of M. leprae proteins
have been identified, enabling us to get more insight into the
pathogenesis of leprosy. We have recently identified an M. leprae gene that is homologous to thioredoxin (Trx) ()and
thioredoxin reductase (TR). Unlike in other organisms, in M. leprae Trx and TR are expressed as a single protein, of which the N
terminus is homologous to the TR and the C terminus to Trx. The two
proteins, when compared to TR and Trx of Escherichia
coli(3, 4) , are fused via a peptide spacer of 22
amino acids(5) . In other organisms, Trx and TR, together with
NADPH, form a redox complex in which TR catalyzes the electron
transport from NADPH to Trx. The reduced thioredoxin then functions as
an electron donor in a wide variety of different metabolic processes (6, 7) . In this study we describe the biological
activity of the M. leprae TR/Trx protein that was obtained by
overexpression in E. coli and subsequent purification. Its
activity is compared with that of separately expressed recombinant M. leprae thioredoxin and thioredoxin reductase.
For overexpression of the TR part of the protein, two stop codons were introduced in the gene using a reversed PCR primer with two stop codons and a HindIII site. The forward primer annealed upstream of the internal EcoRI site. The PCR fragment digested with EcoRI and HindIII was subcloned in pIC/537 creating pIC/TR and subsequently subcloned in pTrcHIS-A creating pHIS/TR.
Subcloning the part of the gene coding for Trx was done by PCR where the forward primer contained a BamHI site and the reversed primer contained a HindIII site. The digested PCR product was subcloned in pTrcHIS-A creating pHIS/Trx. The nucleotide sequence of all cloned PCR products was established for verification.
Prolonged induction at lower temperatures was used to avoid the accumulation of insoluble aggregates that were found in the TR and TR/Trx recombinants when cultured at higher temperatures (e.g. 37 °C). The accumulation of insoluble aggregates is in conflict with the findings of LaVallie et al.(13) . In their now commercially available Trx fusion system, a protein of interest is fused to the C terminus of Trx, and the fusion protein is supposed to remain in the soluble fraction and is released upon osmotic shock. When the naturally occurring M. leprae TR/Trx hybrid protein was overexpressed in E. coli by inducing for 4 h at 37 °C, most of the protein is found in inclusion bodies and could not be osmotically released. An explanation for these findings could be that in our hybrid protein the N terminus of Trx is fused at the C terminus of thioredoxin reductase, whereas in the Trx expression system the C terminus of Trx is fused to the N terminus of the protein of interest. The part of the M. leprae gene encoding Trx overexpressed in E. coli remains soluble.
Fig. 1shows the expression of Trx, TR, and TR/Trx by the recombinant strains before (lanes 1) and after over-night induction at 22 °C (lanes 2). Lanes 3 shows the proteins after purification on a nickel affinity column.
Figure 1:
SDS-polyacrylamide gel electrophoresis
analysis of the overexpressed and affinity-purified proteins. Lanes
1 are before induction, lanes 2 are after induction of
the protein with isopropyl-1-thio--D-galactopyranoside,
and lanes 3 are after purification on the nickel affinity
column. M is the molecular weight marker; molecular mass is
indicated in kDa.
Figure 2:
Redox activity of the M. leprae hybrid protein compared with the separately expressed M.
leprae TR and Trx. The hybrid protein () and the separately
expressed TR + Trx (
) were tested in the insulin reduction
assay using different equimolar concentrations of 1-4 µM (A). B shows the clear difference in activity
between the two systems in the low concentration
range.
Figure 3:
Effect of the addition of exogenous Trx to
TR/Trx compared to TR. In the insulin reduction assay, Trx was added in
a concentration range of 0.1-6.4 µM to fixed amounts
of TR/Trx (, 0.5 µM;
, 1 µM)
and TR (
, 0.5 µM;
, 1
µM).
Figure 4:
Effect of the addition of exogenous TR to
TR/Trx. A concentration range of Trx () or TR (
) of
0.1-6.4 µM was added to a fixed amount of TR/Trx
(0.1 µM), and the effect of both proteins on the hybrid
was tested in the insulin reduction assay.
Taken together, these data indicate that the Trx of the hybrid protein can accept electrons from exogenous TR, whereas TR of the hybrid protein can use exogenous Trx to donate electrons to. Therefore, the enzyme activity of the TR/Trx hybrid protein appears to involve both intramolecular and intermolecular interactions.
The results in this paper show that the M. leprae protein TR/Trx, which is a thioredoxin and thioredoxin reductase hybrid protein, is functionally active. Comparison of the thioredoxin reductase part of the protein with that of E. coli shows that it is elongated at the C terminus by 22 amino acids that continue into the start of thioredoxin. Apparently, this 22-amino acid spacer gives the protein enough flexibility to permit interaction between the TR and Trx entities of this protein. Alternatively, the spacer does not inhibit intermolecular interactions of either the TR or the Trx part of the hybrid protein with free Trx and TR, respectively.
So far no crystals of the TR enzyme complexed with Trx have been obtained. A crystal structure of the reduced state of this M. leprae TR/Trx hybrid protein might shed more light on the interaction between the two subunits.
Combining thioredoxin and thioredoxin reductase in one hybrid protein may be of advantage when the intracellular concentration is low and intramolecular interactions are favored. M. leprae resides inside macrophages, where it has to deal with strongly triggered oxidative burst. Because Trx has been shown to reduce reactive oxygen species(16) , this redox hybrid system might contribute to the escape mechanisms of the bacterium. We have preliminary results that indicate that the TR/Trx protein can exert its function outside the bacterial cell in the macrophage environment where the linkage of TR to Trx in a hybrid form might be particularly advantageous. We are currently investigating how M. leprae benefits from this unique redox system.