Characterization of the N-Acetyl-alpha-D-glucosaminyl L-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis

Center for Structural Biology, Wake Forest University School of Medicine,Winston-Salem, North Carolina 27157, USA.
Biochemistry (Impact Factor: 3.01). 09/2010; 49(38):8398-414. DOI: 10.1021/bi100698n
Source: PubMed

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-hydroxy acids, as the 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. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the 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.

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    • "Note that fosfomycin resistance provides a semiquantitative measure of in vivo BSH levels since resistance is largely dependent on the BSH-dependent thiol-transferase FosB. Since the K m of FosB for BSH is in the millimolar range (higher than in vivo levels during growth; Roberts et al., 2013), FosB activity, and therefore fosfomycin resistance, is very sensitive to even small changes in BSH levels (Gaballa et al., 2010; Parsonage et al., 2010). Since pMUTIN disruptants are generally polar on downstream genes (Fig. 3c), this is suggestive of another promoter directly upstream of bshC. "
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    ABSTRACT: Bacillithiol is the major low molecular weight thiol produced by many Firmicutes bacteria, including the model organism Bacillus subtilis and pathogens such as B. anthracis and Staphylococcus aureus. We have previously shown that four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical σA-dependent promoters as determined by 5’RACE. The bshA and bshB1 genes are embedded within a seven gene operon additionally including mgsA, encoding methylglyoxal synthase, and the essential genes cca and birA, encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The bshB2 gene is co-transcribed with unknown function genes, while bshC is expressed both as part of a two gene operon (with the upstream putative pantothenate biosynthesis gene ylbQ) as well as from its own promoter. All three operons are expressed at a reduced level in an spx null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox center of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an spx null mutant.
    Microbiology 07/2013; DOI:10.1099/mic.0.070482-0. · 0.71 Impact Factor
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    • "In 2008, Ruane et al. reported the crystal structure of a putative glycosyltransferase, ORF BA1558, which is the Bacillus anthracis homolog of BshA [5]. Parsonage et al. reported the structure of this B. anthracis BshA with UDP-malate ternary complex and described the phenotype of the mutant disrupted in this gene [6]. Herein, we report on the characterization of B. subtilis BshA and Staphylococcus aureus BshA and show that BSH is able to inhibit BshA. "
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    ABSTRACT: The first step during bacillithiol (BSH) biosynthesis involves the formation of N-acetylglucosaminylmalate from UDP-N-acetylglucosamine and l-malate and is catalyzed by a GT4 class glycosyltransferase enzyme (BshA). Recombinant Staphylococcus aureus and Bacillus subtilis BshA were highly specific and active with l-malate but the former showed low activity with d-glyceric acid and the latter with d-malate. We show that BshA is inhibited by BSH and similarly that MshA (first enzyme of mycothiol biosynthesis) is inhibited by the final product MSH.
    FEBS letters 04/2012; 586(7):1004-8. DOI:10.1016/j.febslet.2012.02.028 · 3.34 Impact Factor
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    • "D. A Specific Example: Flexibility of the BcZBP Deacetylase BcZBP is a zinc-dependent deacetylase from Bacillus cereus whose crystal structure has been reported at the resolution of 1.8 A ˚ (Fadouloglou et al., 2006, 2007). The biological pathway and the function of the protein were unknown until recently when the ortholog from Bacillus anthracis BaBshB (with 97% sequence identity) was identified as a deacetylase involved in the bacillithiol biosynthesis (Newton et al., 2009; Parsonage et al., 2010). BcZBP is a hexamer and possesses six structurally equivalent active sites which are formed by the association of two monomers. "
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    ABSTRACT: The dynamic nature of protein structures has been recognized, established, and accepted as an intrinsic fundamental property with major consequences to their function. Nowadays, proteins are considered as networks of continuous motions, which reflect local flexibility and a propensity for global structural plasticity. Protein-protein and protein-small ligand interactions, signal transduction and assembly of macromolecular machines, allosteric regulation and thermal enzymatic adaptation are processes which require structural flexibility. In general, enzymes represent an attractive class among proteins in the study of protein flexibility and they can be used as model systems for understanding the implications of protein fluctuations to biological function. Flexibility of the active site is considered as a requirement for reduction of free energy barrier and acceleration of the enzymatic reaction while there is growing evidence which concerns the connection between flexibility and substrate turnover rate. Moreover, the role of conformational flexibility has been well established in connection with the accessibility of the active site, the binding of substrates and ligands, and release of products, stabilization and trapping of intermediates, orientation of the substrate into the binding cleft, adjustment of the reaction environment, etc.
    Advances in Protein Chemistry and Structural Biology 01/2012; 87:181-218. DOI:10.1016/B978-0-12-398312-1.00007-X · 3.74 Impact Factor
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