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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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    • "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.
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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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