Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-11289]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for † This work was supported by National Institutes of Health (NIH) Grants GM-35394 (A.C), AI-49174 (R.C.F.), and GM-62896 (S.J.), by a grant from the Southeast Regional Center of Excellence for Biodefense and Emerging Infections (SERCEB, to A.C.), and by Cancer Center (CORE) Support Grant CA21765 and ALSAC (S.J.). C.P. was the recipient of a Graduate Fellowship from the U.S. Department of Homeland Security (DHS). SERCEB is supported by an award from the NIH (National Institute of Allergy and Infectious Diseases; NIAID). The DHS Scholarship and Fellowship Program is administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement with the U.S. Department of Energy (DOE). ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-AC05-06OR23100. The findings, opinions, and recommendations expressed in this paper are those of the authors and are not necessarily those of NIAID, SERCEB, NIH, DHS, DOE, or ORISE. Data for this study were measured at beamline X12C of the National Synchrotron Light Source. With the identification of two distinct new bacterial pantothenate kinase classes in 2005, different investigators have used either type I, -II, and -III CoaAs (e.g., type II Staphylococcus aureus CoaA) or PanK-I, -II, and -III nomenclature to identify the three bacterial enzyme classes. In this report we refer to the three bacterial enzyme classes as bacterial type I, type II, and type III PanKs; CoaA is synonymous with the type I PanK. 5 J. Ravel, personal communication. Supporting Information Available A figure depicting a structure-based alignment of 13 type III PanK sequences ( Figure S1) is included as Supporting Information (one page). 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 2009 January 5. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 Å, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to prov...

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis [Nicely, N.I., Parsonage, D., Paige, C., Newton, G.L., Fahey, R.C., Leonardi, R., Jackowski, S., Mallett, T.C., and Claiborne, A. (2007) Biochemistry 46, 3234-3245], and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Å resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 Å resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2′-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367′-OH and Tyr425′-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425′ is the primary proton donor in catalysis, with Tyr367′ functioning as a cryptic alternate donor in the absence of Tyr425′.

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...

SummaryGranuloma formation represents a pivotal point during human infection with Mycobacterium tuberculosis, for this structure may limit mycobacterial spread and prevent active disease, while at the same time allow for the survival and persistence of viable mycobacteria within the host. The current therapeutic regimens for treating tuberculosis disease have proven effective in developing countries. However, in countries with large populations, limited access to health care, and high incidence of HIV co-infection, tuberculosis disease continues to represent a major global health emergency. Particularly, the emergence of extensively and multidrug-resistant forms of tuberculosis underscores the need develop new treatment strategies. Recent mechanistic studies have identified bacterial virulence mechanisms that subvert host responses and lead to an inappropriate upregulation of host factors such as tumour necrosis factor-a (TNF-a) and matrix metalloproteinases (MMPs). Paradoxically, then, part of the mycobacterial virulence programme may be to promote granuloma development and maturation. These observations suggest that together with appropriate anti-microbials host-based therapeutics directed at TNF-a and MMP inhibition may counteract the microbial subterfuge, reduce the pro-granulomatous response, and offer an enhanced therapeutic effect. Hostdirected therapy that alters the immune response may offer an alternative approach towards reducing treatment duration, the risk of anti-microbial resistance and improving patient outcome.

In Bacillus anthracis, the novel type III pantothenate kinase (PanK Ba ; encoded by coaX) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional coaX mutant that PanK Ba is an essential enzyme, thus contributing to its validation as a new antimicrobial target.Coenzyme A (CoASH) (20) is the major low-molecularweight thiol in Bacillus anthracis (25); the tripeptide thiol glutathione (8) is absent in all species of Bacillus analyzed to date (23,24,32 (25) have demonstrated a strong correlation among the absence of glutathione biosynthesis, the absence of the type I PanK, and the presence of the type III enzyme for 14 phylogenetic classes of bacteria.In their transcriptional profiling of the B. anthracis life cycle, in which five distinct temporal waves of gene expression were identified from germination through sporulation, Bergman et al. (4) showed that the coaX, coaBC (BA4007), and coaD (BA4139) genes encoding the first three enzymes in the Pan3CoASH pathway (20) are upregulated in waves I and II. The coaX gene was also upregulated more than twofold between 1 and 2 h postinfection within host macrophages (3); in this respect (4), PanK Ba may be particularly significant as a potential target for the design of therapeutically useful molecules. CoASH biosynthesis has very recently been reviewed as an antimicrobial drug target (31); crystal structures are now available for PanK Ba (25) and the type III PanKs from Pseudomonas aeruginosa (15) and Thermotoga maritima (35, 36), and they include complexes with Pan and ADP and with the 4Ј-phosphopantothenate product. These have led to the identification of new motifs for the Pan-binding pocket and suggest, based on differences in the binding modes for both Pan and ATP substrates (relative to the type II human PanK) (14), potential modes of design for new inhibitors specifically targeting the type III enzymes.As suggested by the absence of glutathione, CoASH also functions in the thiol-disulfide redox biology of B. anthracis (25); this scheme includes an NAD(P)H-dependent CoA-disulfide reductase (BACoADR) that maintains the reduced intracellular state of CoASH in vegetative cells via an enzymatic . This is quite distinct from the system described for B. subtilis, which has both type I and type III PanKs (6, 37) but lacks CoA-disulfide reductase (34). While the recent report of Hochgräfe et al. (13) supports the conclusion that S-thiolation by Cys represents a general, reversible mechanism for the protection of protein-SH groups during disulfide stress in B. subtilis, earlier studies with Bacillus megaterium linked CoA-disulfide reductase (32) with the reduction of spore protein-SSCoA (soluble proteins S-thiolated with CoASH) early in germination. These protein-SSCoA mixed disulfide forms account for ϳ45% of the total CoASH in B. megaterium spores (30).Bioinformatics analyses of the PanK Ba locus (26) indicated that coaX might be linked with the hslO and cysK-1 genes (BA0066 and BA0067, r...

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