Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis .

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, 5-7) have NAD(P)H-dependent coenzyme A-disulfide reductases (CoADRs).1 We have also shown that the type III pantothenate kinase, which is unusual in its insensitivity to feedback inhibition by CoASH (2), is essential for growth of B. anthracis (8). CoADR, the chimeric CoADR-RHD protein (9), and the type III pantothenate kinase represent three well-characterized adaptations to the CoASH-based redox buffer system for this Gram-positive pathogen. A new unknown thiol, originally referred to as U12, was also identified in the B. anthracis extract (2); a mass of 398 Da was reported for U12-SH. By combining analytical chemical approaches with mass spectrometry and NMR, the structure of U12 has been determined to be N-cysteinyl-α-D-glucosaminyl L-malate, and U12 has been renamed bacillithiol [BSH (10)]. BSH is present at intracellular concentrations of 0.1-0.35 mM 2 in a number of Bacillus species, including B. anthracis, Bacillus subtilis, and Bacillus megaterium, as well as in S. aureus. While none of the four B. subtilis strains represented in the NCBI database have orthologs of CoADR or any of its isoforms (9), B. anthracis, B. megaterium (11), and S. aureus (3) maintain both CoASH and BSH redox buffer systems, in the absence of GSH. Pother et al. (12), in a recent study with S. aureus demonstrating that the majority of reversible protein thiol oxidations observed during treatment of cells with diamide are based on S-thiolations with Cys rather than protein disulfide formation, have concluded that Cys also functions as an important thiol redox buffer in S. aureus. In an initial study of the biosynthesis and functions of BSH (13), we have demonstrated that the B. subtilis YpjH protein (BsuBshA), the enzyme orthologous to B. anthracis ORF BA1558, catalyzes the synthesis of N-acetyl-α-D-glucosaminyl L-malate (GlcNAc-malate) from UDP-GlcNAc and L-malate; the product structure has been confirmed by mass spectrometry (10, 13). We have also demonstrated that the BA1557 gene product (BaBshB) catalyzes the deacetylation of GlcNAc-malate (13), providing the free 2-amino group of the GlcN moiety required for the Cys ligation step that is proposed to complete the synthesis. Deletion of the B. subtilis yllA locus, predicted to be involved in the biosynthetic pathway and initially suggested to encode this putative ligase, does eliminate BSH production; the recombinant protein, however, does not catalyze ATP-dependent BSH formation from Cys and GlcN-malate, as assayed in vitro. In the absence of functional information, Ruane et al. (14) have reported the structure of the ORF BA1558 (identified in this work as BaBshA) apoenzyme, refined at a resolution of 3.1 A. Although this enzyme, like the MshA glycosyltransferase that produces GlcNAc-Ins-P in the first step of mycothiol (MSH) biosynthesis (15, 16), is a member of the GT-B and GT4 fold (17) and Carbohydrate-Active enZymes (18) families, respectively, structural homology searches using the MshA apoenzyme structure were unsuccessful in identification of other GT-B family members, including ORF BA1558. This indicates a major conformational difference between the two apoenzymes, despite the similarity in reactions catalyzed. Fadouloglou et al. (19) reported the crystal structure of the Zn2+-binding BcZBP protein from Bacillus cereus, as refined at a resolution of 1.8 A. BcZBP is the enzyme orthologous to BaBshB, the GlcNAc-malate deacetylase, with 97% sequence identity. A recent kinetic analysis (20) with GlcNAc and several GlcNAc oligomers indicated that, although the biological function and natural substrate for BcZBP were unknown, the enzyme had optimal activity with (GlcNAc)2 (kcat/Km = 3.3 × 107 M-1s-1 at 37°C). The enzyme is a member of the PIG-L superfamily represented by GlcNAc-phosphatidylinositol deacetylase (21), the MshB deacetylase (22) of the MSH biosynthetic pathway, and the mycothiol S-conjugate amidase (23). The active-site Zn2+ of BcZBP is buried at the bottom of a ca. 12 A deep cavity on the hexamer surface, and Arg140 is prominently positioned at the entry of the cavity. Both R140A and R140E mutants exhibit kcat/Km values 0.2-0.3% that of wild-type enzyme (20). His12, Asp15, and His113 provide the protein ligands to the Zn2+, and Asp14 has been proposed as an acid-base catalyst in the hydrolase reaction. A model of a BcZBP-GlcNAc complex reveals that this substrate is considerably smaller than the volume of the cavity; a hydrophilic patch consisting of the side chains of Asp108, Asn150, and Tyr194 marks an unoccupied region proximal to the Zn2+. The Y194F mutant specificity constant, which is <0.1% that of wild-type (20), reflects a dominant effect on Km. In particular, the anomeric C1-OH of GlcNAc also appears to be accommodated by a relatively large space within the cavity. For the BcZBP apoenzyme structure, three very mobile loops have recently been implicated in determining active-site accessibility and regulating substrate specificity (24). Given the goal of better defining the thiol redox buffer in B. anthracis and characterizing potential new targets for the development of antimicrobial agents that are selective against B. anthracis, S. aureus, and other Gram-positive pathogens that rely on the BSH-based redox buffer system, we report here detailed kinetic analyses of the BaBshA glycosyltransferase and BaBshB deacetylase. The kinetic studies are interpreted in view of the ORF BA1558 and BcZBP crystal structures, and we also provide the structure of the BaBshA-UDP-malate complex, refined at a resolution of 3.3 A, which demonstrates the absence of any major conformational change on ligand binding. In addition we show that deletion of the BAS1445 locus encoding BshA in B. anthracis Sterne generates a BSH-deficient strain appropriate for testing in a murine model of inhalational anthrax.