BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N -acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis , BA1557 has been identified as the N -acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆ BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N -acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH–activity profile and Zn 2+ -dependent catalytic acid–base reaction provides further evidence for their cross-functionalities.

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus ( Sa FosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. Sa FosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K m values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for Sa FosB recognition and catalytic efficiency. Metal activity assays indicated that Mn 2+ and Mg 2+ are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of Sa FosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS–fosfomycin), this demonstrates that the Sa FosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that Sa FosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus .