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Structure of the type III pantothenate kinase from Bacillus anthracis at 2.0 A resolution: implications for coenzyme A-dependent redox biology.

ABSTRACT: 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, 11278-89]. 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 the conversion of pantothenate (Pan) to CoASH in Escherichia coli are conserved in B. anthracis. 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 A, 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 providing a clear rationale for the absence of CoASH inhibition, analysis of the Pan-binding pocket has led to the development of two new structure-based motifs (the PAN and INTERFACE motifs). Our analyses also suggest that the type III PanK in the spore-forming B. anthracis plays an essential role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

SUBMITTER: Nicely NI

PROVIDER: S-EPMC2613803 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Identification of Specific and Nonspecific Inhibitors of Bacillus anthracis Type III Pantothenate Kinase (PanK).

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2004-01-08 | GSE840 | GEO

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: comparison with isoform G and implications for structure-based drug discovery.

Project description:Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises approximately 530 residues, whereas isoform G, found only in bacteria, is larger by approximately 200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 A resolution) and in complex with acetyl-coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 A resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 A resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted alpha/beta domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.

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Pyridine nucleotide complexes with Bacillus anthracis coenzyme A-disulfide reductase: a structural analysis of dual NAD(P)H specificity.

Project description: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 A 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 A 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'.

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