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.
Wieles
B
B
Department of Immunohematology, Leiden University Hospital, The Netherlands.
van Noort
J
J
Drijfhout
J W
JW
Offringa
R
R
Holmgren
A
A
Ottenhoff
T H
TH
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D009166:Mycobacterium leprae / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D001426:Bacterial Proteins; D004220:Disulfides; D007328:Insulin; D011994:Recombinant Proteins; C096391:thioredoxin-thioredoxin reductase hybrid protein, Mycobacterium leprae; D013879:Thioredoxins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase