Bacillithiol is a low-molecular-weight thiol antioxidant found in Firmicutes like the pathogens Staphylococcus aureus and Bacillus subtilis. It performs a key role in these organisms’ response to oxidative stress. Specifically, bacillithiol is used for detoxifying free oxidative species within the cell or for S-bacillithiolation of redox-sensitive cysteine residues of proteins. S-bacillithiolation protects sensitive residues from overoxidation with a potential function as a regulator of enzyme activity. The glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) demonstrates a reversible decrease in activity when S-bacillithiolated. This serves as a potential mechanism of redirecting metabolism through the pentose phosphate pathway to generate reducing power in response to oxidative stress. When oxidative stress is no longer present, bacillithiol must be removed to revert proteins to their unmodified state. The enzyme BrxA removes bacillithiol from redox-sensitive cysteine residues as part of the debacillithiolation system. BrxA has been shown to have a redox-dependent quaternary structure favoring a homodimer under oxidizing conditions and a monomer under reducing conditions; however, the implications of dimerization on enzyme activity are still unclear. Our analysis of the effects of BrxA oligomerization on its function is presented. The redox-sensitive responses of BrxA and GAPDH demonstrated through enzymatic activity assays in addition to novel X-ray crystallographic structures suggest that the debacillithiolation system may be regulated by the redox environment as a way of responding to oxidative stress within the cell. Mechanistically defining BrxA’s role in debacillithiolation is key to understanding the response of Firmicute pathogens to oxidative stresses like those presented by the human immune system.

Paul Cook

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