Gaballa, Ahmed, Chi, Bui Khanh, Roberts, Alexandra A., Becher, Dörte, Hamilton, Chris J., Antelmann, Haike, and Helmann, John D. (2014) Redox regulation in Bacillus subtilis: the bacilliredoxins BrxA(YphP) and BrxB(YqiW) function in de-bacillithiolation of S-bacillithiolated OhrR and MetE. Antioxidants & Redox Signaling, 21 (3). pp. 357-367.

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Abstract

Aims: In bacillithiol (BSH)-utilizing organisms, protein S-bacillithiolation functions as a redox switch in response to oxidative stress and protects critical Cys residues against overoxidation. In Bacillus subtilis, both the redox-sensing repressor OhrR and the methionine synthase MetE are redox controlled by S-bacillithiolation in vivo. Here, we identify pathways of protein de-bacillithiolation and test the hypothesis that YphP(BrxA) and YqiW(BrxB) act as bacilliredoxins (Brx) to remove BSH from OhrR and MetE mixed disulfides.

Results: We present evidence that the BrxA and BrxB paralogs have de-bacillithiolation activity. This Brx activity results from attack of the amino-terminal Cys residue in a CGC motif on protein BSH-mixed disulfides. B. subtilis OhrR DNA-binding activity is eliminated by S-thiolation on its sole Cys residue. Both the BrxA and BrxB bacilliredoxins mediate de-bacillithiolation of OhrR accompanied by the transfer of BSH to the amino-terminal cysteine of their CGC active site motif. In vitro studies demonstrate that BrxB can restore DNA-binding activity to OhrR which is S-bacillithiolated, but not to OhrR that is S-cysteinylated. MetE is most strongly S-bacillithiolated at Cys719 in vitro and can be efficiently de-bacillithiolated by both BrxA and BrxB.

Innovation and Conclusion: We demonstrate that BrxA and BrxB function in the reduction of BSH mixed protein disulfides with two natural substrates (MetE, OhrR). These results provide biochemical evidence for a new class of bacterial redox-regulatory proteins, the bacilliredoxins, which function analogously to glutaredoxins. Bacilliredoxins function in concert with other thiol-disulfide oxidoreductases to maintain redox homeostasis in response to disulfide stress conditions.

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