Bacillithiol is a compound produced by several Gram-positive bacterial species including the human pathogens Staphylococcus aureus and Bacillus anthracis. It is involved in maintaining cellular redox balance as well as the destruction of reactive oxygen species and harmful xenobiotic agents, including the antibiotic fosfomycin. BshA, BshB, and BshC are the enzymes involved in bacillithiol biosynthesis. BshA is a retaining glycosyltransferase responsible for the first committed step in bacillithiol production, namely the addition of N-acetylglucosamine to l-malate. Retaining glycosyltransferases like BshA are proposed to utilize an SNi-like reaction mechanism in which leaving group departure and nucleophilic attack occur on the same face of the hexose. However, significant questions persist regarding the details of how BshA and similar enzymes accommodate their substrates and facilitate catalysis. Here we report X-ray crystallographic structures of BshA from Bacillus subtilis 168 bound with UMP and/or GlcNAc-mal at resolutions of 2.15 and 2.02 Å, respectively. These ligand-bound structures, along with our functional and computational studies, provide clearer insight into how BshA and other retaining GT-B glycosyltransferases operate, corroborating the substrate-assisted, SNi-like reaction mechanism. The analyses presented herein can serve as the basis for the design of inhibitors capable of preventing bacillithiol production and subsequently, help combat resistance to fosfomycin in various pathogenic Gram-positive microorganisms.

Bacillithiol is produced by many Gram-positive bacteria via a pathway utilizing the enzymes BshA, BshB, and BshC. Here we report the 1.77 Å resolution crystal structure of BshC, the putative cysteine ligase in bacillithiol production. The structure reveals that BshC contains a core Rossmann fold with connecting peptide motifs (CP1 and CP2) and a unique α-helical coiled-coil domain that facilitates dimerization. The model contains citrate and glycerol in the canonical active site and ADP in a second binding pocket. The overall structure and bound ligands give insight into the function of this unique enzyme.

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Bacillithiol (BSH) is the major low molecular weight thiol in many gram-positive organisms. This compound is involved in the detoxification of xenobiotic compounds, the maintenance of redox homeostasis, and is the preferred cosubstrate for the fosfomycin resistance enzyme FosB. Organisms with knocked-out BSH biosynthesis genes have demonstrated an increased sensitivity to fosfomycin, making BSH production a logical target for inhibition. In the first committed step of the BSH biosynthesis pathway, the enzyme BshA catalyzes the formation of N-acetylglucosaminylmalate (GlcNAc-Mal) via an activated sugar donor and l-malate acceptor. BshA is a GT-B retaining glycosyltransferase and likely utilizes an SNi-like retaining mechanism. To further characterize BshA and other GT-B retaining glycosyltransferases, we embarked on a structural and functional analysis of BshA. Our lab has determined several X-ray crystallographic structures of wild-type BshA from Bacillus subtilis with products bound. In addition, we have determined an initial structure of the S17A mutant of BshA from Bacillus subtilis that contains the substrate UDP-N-acetylglucosamine bound within the active site. These structures provide compelling evidence for the proposed SNi-like mechanism. We also report progress on the structural and functional analysis of the BshA enzyme from Staphylococcus saprophyticus. Together, these analyses further our understanding of BshA and other GT-B retaining glycosyltransferases, which may be used in the future to model BshA inhibitors and increase the effectiveness of fosfomycin antibiotics.

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