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′.