Characterization of the N-Acetyl-α-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis,

Abstract

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular-weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other α-hydroxyacids, as acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. SUPPORTING INFORMATION AVAILABLEFigures S1 and S2, depicting preparative-scale production and NMR characterization of GlcNA-cmalate, Figure S3, providing reciprocal plots of BaBshA activity, and Tables S1 and S2, providing the primer sequences used in the study and describing the acceptor substrate specificity for BaBshA. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis B. anthracis bshA locus (BA1558) has been identified in a transposon site hybridization study as required for growth, sporulation, or germination, suggesting that the biosynthesis of BSH could represent a target for development of novel antimicrobials with broad spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.In his recent review on the management of oxidative stress in Bacillus, Zuber (1) concludes that the metabolites that function in redox buffering and thiol homeostasis, and their influence on the oxidative stress response, are not well understood. Earlier work from this laboratory (2) demonstrated that CoASH provided the major low-molecular weight thiol redox buffer in Bacillus anthracis, replacing GSH as had previously been demonstrated for Staphylococcus aureus (3). The likelihood that CoASH plays an important functional role in redox buffering and thiol homeostasis is strengthened by the demonstration that both B. anthracis (4) and S. aureus (3...

S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics

“…The observed Scysteinylations could originate from S-bacillithiolations that could be explained by the BSH biosynthesis pathway. The pathways for BSH biosynthesis and degradation have been identified in B. subtilis and the structures of the L-malic acid glycosyltransferase BaBshA and the GlcNAc-Mal deacetylase BaBshB have been resolved in B. anthracis (35,90). There are two GlcNAc-Mal deacetylases in B. subtilis, BshB1 and BshB2 with similar activities.…”

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

“…The first gene of the BSH biosynthetic pathway, bshA, is responsible for conjugating L-malic acid to UDP-GlcNAc to form N-acetylglucosaminylmalate and is necessary for the synthesis of BSH (11,12). The next gene, bshB, encodes an enzyme responsible for deacetylating GlcNAc-Mal, and the last gene, bshC, codes for a cysteine ligase (11).…”

“…The next gene, bshB, encodes an enzyme responsible for deacetylating GlcNAc-Mal, and the last gene, bshC, codes for a cysteine ligase (11). While multiple bshB paralogs occur in Bacillus species (11,12), there is only one, bshB2, in S. aureus (11). Interestingly, laboratory strains of S. aureus in the 8325 lineage, such as SH1000 and 8325-4, contain an 8-bp insertion in bshC which renders the protein inactive and, hence, prevents the synthesis of BSH (13,14).…”

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

“…BSH appears to participate in sensing of peroxides and may substitute for GSH, but gene knock-outs abolishing BSH synthesis in both B. subtilis (22) and B. anthracis (23) are viable without obvious growth defects. The absence of BSH does however affect sporulation efficiency in both bacteria as well as tolerance against high salt and low pH in B. subtilis (22,23).…”