Functional studies of multiple thioredoxins from Mycobacterium tuberculosis

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• The thioredoxin superfamily mediates cytoplasmic protein reduction and maintains cellular redox homeostasis.
• Mycobacterium tuberculosis possesses three potential thioredoxin genes: trxAMtb, trxBMtb, and trxCMtb.

Purpose of the Study:

• Characterize the thioredoxin system of Mycobacterium tuberculosis.
• Determine the biological activity and redox properties of M. tuberculosis thioredoxins.

Main Methods:

• Overproduction of thioredoxins in Escherichia coli.
• Assay of thioredoxin reductase activity using insulin as a substrate.
• Measurement of redox potentials.
• Analysis of mRNA transcripts under various growth conditions.
• Heterologous expression in E. coli deficient strains.

Main Results:

• All three M. tuberculosis thioredoxins (TrxAMtb, TrxBMtb, TrxCMtb) reduced insulin in vitro.
• Only TrxBMtb and TrxCMtb were reduced by thioredoxin reductase in an NADPH-dependent manner, indicating biological activity.
• TrxAMtb expression appears cryptic due to absent mRNA transcripts.
• TrxBMtb and TrxCMtb exhibit redox potentials slightly more oxidizing than E. coli TrxA.
• Mycobacterial thioredoxins functionally substituted for E. coli cytoplasmic reduction pathways.

Conclusions:

• TrxBMtb and TrxCMtb are the active disulfide reductases in the M. tuberculosis thioredoxin system.
• TrxAMtb may have a cryptic or regulated function.
• The M. tuberculosis thioredoxin system contributes to redox homeostasis and can function in heterologous systems.