Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

Abstract

Background Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. Methods We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. Results BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. Conclusions BstA is a bacillithiol transferase from Staphylococcus aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. General Significance BstA is an active bacillithiol transferase from Staphylococcus aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from Bacillus subtilis YfiT.

Bacillithiol: a key protective thiol inStaphylococcusaureus

“…The native substrates for two mycothiol-dependent enzymes from the S -transferase like (STL) superfamily [52], formerly known as the DinB/YfiT-like superfamily, have been characterized from Actinomycetes. Corynebacterium glutamicum was found to grow on aromatic hydrocarbons like gentisate as a sole carbon source in a mycothiol-dependent manner.…”

“…The single predicted S. aureus Newman STL enzyme (ORF ID NWMN_2591) was identified in a Superfamily search (http://supfam.org/SUPERFAMILY/) using B. subtilis BstA as a query sequence against the S. aureus Newman genome. The S. aureus enzyme was confirmed to be a bacillithiol transferase on the basis of biochemical studies of cell free extracts [22] and the purified protein and was named S. aureus BstA [52]. Kinetic studies with S. aureus BstA demonstrated that the K m for BSH is 16 ± 4 µM, which is ~10-fold lower than the intracellular concentration of bacillithiol in S. aureus , indicating that the enzyme is saturated with bacillithiol in vivo [52].…”

“…The S. aureus enzyme was confirmed to be a bacillithiol transferase on the basis of biochemical studies of cell free extracts [22] and the purified protein and was named S. aureus BstA [52]. Kinetic studies with S. aureus BstA demonstrated that the K m for BSH is 16 ± 4 µM, which is ~10-fold lower than the intracellular concentration of bacillithiol in S. aureus , indicating that the enzyme is saturated with bacillithiol in vivo [52]. S. aureus BstA was found to catalyze the addition of bacillithiol to cerulenin [22] at a low rate in vitro [52].…”

“…Kinetic studies with S. aureus BstA demonstrated that the K m for BSH is 16 ± 4 µM, which is ~10-fold lower than the intracellular concentration of bacillithiol in S. aureus , indicating that the enzyme is saturated with bacillithiol in vivo [52]. S. aureus BstA was found to catalyze the addition of bacillithiol to cerulenin [22] at a low rate in vitro [52]. This newly identified STL superfamily is a diverse protein family that contains ~30,000 proteins with structural similarity but very little sequence identity.…”

“…S. aureus contains only one BshB2-type enzyme and the bshB NARSA transposon mutant (SAUSA300_0552) was determined by thiol analysis to be a complete BSH knockout [22,52]. The same bshB transposon mutant also lacks 90% of the wildtype bacillithiol S -conjugate amidase activity, indicating that this protein is responsible for all of the BshB activity and the majority of bacillithiol conjugate amidase activity in S. aureus [52].…”