Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Abstract

Bacillithiol (BSH) is the major low-molecular-weight (LMW) thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa, redox potential and thiol–disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mm, which is significantly greater than previously observed from single measurements taken during mid-exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.

Structural and Biochemical Characterizations of Methanoredoxin from Methanosarcina acetivorans, a Glutaredoxin-Like Enzyme with Coenzyme M-Dependent Protein Disulfide Reductase Activity

“…45 Bacillithiol is a glutathione surrogate of Bacillus subtilis and other firmicutes. 46,47 Thus, it is not surprising that CoMSH functions as a thiol protectant in M. acetivorans and doubtless in other phylogenetically and metabolically diverse methanogens where CoMSH is universal. It follows that CoMSH and MRX are expected to play important roles in redox control, signaling, and oxidative stress similar to GSH and GRXs of the domain Bacteria.…”

Redox Regulation in Bacillus subtilis: The Bacilliredoxins BrxA(YphP) and BrxB(YqiW) Function in De-Bacillithiolation of S-Bacillithiolated OhrR and MetE

“…3B). Nevertheless, BSH (rather than free Cys) is the major cofactor for mixed disulfide formation in vivo, likely due to the increased thiol acidity of BSH whose reactive thiolate anion concentrations are 10-50-fold higher than those of free Cys thiolate at physiological pH in B. subtilis (34). The addition of DTT to either OhrR-SSCys or OhrR-SSB rapidly and fully restored OhrR DNA-binding activity (Fig.…”

“…This is consistent with the presence in the cell of numerous other thiol-disulfide oxidoreductases, including the essential thioredoxin (Trx) system, and the general notion that these systems are, at least, partially redundant in function. Interestingly, the standard thiol redox potentials of BSH ( -221 mV) (34) and BrxA ( -118 mV) (8) are notably higher than those of GSH ( -240 mV) and typical Grx proteins ( -200 mV) (3). It will be interesting to see whether these biophysical differences between the BSH and GSH redox systems are counterbalanced by differences in the relative kinetic properties of Brx and Grx enzymes.…”

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

“…We also observed rapid, BstA-independent BSH conjugate formation with the thiol reactive molecules 4-hydroxynonenal, sulforaphane, and etacrynic acid (Supplementary Table S1). These results provide evidence for the reactive nature of BSH in conjugation reactions with halides, isothiocyanates, enals, enones and epoxides, perhaps due to the low thiol pKa (pKa 7.46) for BSH[32]. This suggests that BSH might be able to directly detoxify many molecules in living cells without requiring a thiol transferase.…”

“…One proposed function of thiol transferases is to lower the thiol pKa of the co-substrate during catalysis[44]. However, the thiol pKa of BSH is more acidic[32] than the pKa values of the other major endogenous LMWTs from S. aureus (cysteine pKa=8.6; coenzyme A pKa=9.6)[45]. Thiol transferases can also function to bring the thiol cofactor in close proximity to the substrate[44], and it is possible that this is the major role played by BstA.…”