Functional studies of multiple thioredoxins from Mycobacterium tuberculosis.
Cytoplasmic protein reduction via generalized thiol/disulfide exchange reactions and maintenance of cellular redox homeostasis is mediated by the thioredoxin superfamily of proteins. Here, we describe the characterization of the thioredoxin system from Mycobacterium tuberculosis, whose genome bears the potential to encode three putative thioredoxins from the open reading frames designated trxAMtb, trxBMtb, and trxCMtb. We show that all three thioredoxins, overproduced in Escherichia coli, are able to reduce insulin, a model substrate, in the presence of dithiothreitol. However, we observe that thioredoxin reductase is not capable of reducing TrxAMtb in an NADPH-dependent manner, indicating that only TrxBMtb and TrxCMtb are the biologically active disulfide reductases. The absence of detectable mRNA transcripts of trxAMtb observed when M. tuberculosis strain H37Rv was cultivated under different growth conditions suggests that trxAMtb expression may be cryptic. The measured redox potentials of TrxBMtb and TrxCMtb (-262+/-2 mV and -269+/-2 mV, respectively) render these proteins somewhat more oxidizing than E. coli thioredoxin 1 (TrxA). In E. coli strains lacking components of cytoplasmic protein reduction pathways, heterologous expression of the mycobacterial thioredoxins was able to effectively substitute for their function.
Akif
Mohd
M
Laboratory of Structural Biology, Centre for DNA Fingerprinting and Diagnostics, Nacharam, Hyderabad 500076, India.
Khare
Garima
G
Tyagi
Anil K
AK
Mande
Shekhar C
SC
Sardesai
Abhijit A
AA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010084:Oxidation-Reduction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D007328:Insulin; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013879:Thioredoxins; D004229:Dithiothreitol