Structure of the Type III Pantothenate Kinase from <i>Bacillus anthracis</i> at 2.0 Å Resolution:  Implications for Coenzyme A-Dependent Redox Biology<sup>,</sup>

Ni, Nicely; Parsonage, Derek; Paige, Carleitta; Gl, Newton; Rc, Fahey; Leonardi, Roberta; Jackowski, Suzanne; Tc, Mallett; Claiborne, Al

doi:10.1021/bi062299p

Cited by 52 publications

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“…1A) [2]. PanKs occur as one of three distinct forms, known as type I, type II and type III PanK enzymes, respectively [17][18][19][20], with only PanK I and PanK II (the subscript denotes the type) showing activity towards both Pan and PantSH [21]. Organisms such as Pseudomonas aeruginosa that have PanK III enzymes cannot salvage CoA from pantetheine [22] and are also resistant to pantothenamideinduced growth inhibition [17,20].…”

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confidence: 99%

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

et al. 2014

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N-substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well-studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide-induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA-and acyl carrier protein-dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanK I , PanK II and PanK III ) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanK I enzymes accept them as substrates, the highly selective PanK III s are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanK II ) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.

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Section: Introductionmentioning

confidence: 99%

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

et al. 2014

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“…In actinomycetes, GSH is replaced with mycothiol (MSH), an N-acetyl-cysteine derivative of the pseudodisaccharide of glucosamine and myo-inositol (Jothivasan & Hamilton, 2008;Newton et al, 2008). Neither GSH nor MSH has been detected in firmicutes and until recently coenzyme-A was thought to be the main LMW thiol in these bacteria (Nicely et al, 2007). Subsequently, a novel LMW thiol was detected in two independent studies (Lee et al, 2007;Nicely et al, 2007), structurally characterized as the aanomeric glycoside of L-cysteinyl-D-glucosamine with Lmalic acid (Newton et al, 2009), and named bacillithiol (BSH) (for reviews, see Fahey, 2013;Helmann, 2011;Van Laer et al, 2013).…”

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confidence: 99%

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx

et al. 2013

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Bacillithiol is the major low molecular mass thiol produced by many firmicutes bacteria, including the model organism Bacillus subtilis and pathogens such as Bacillus anthracis and Staphylococcus aureus. We have previously shown that four genes (bshA, bshB1, bshB2 and bshC) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical s A -dependent promoters as determined by 59RACE (rapid amplification of cDNA ends). The bshA and bshB1 genes are embedded within a seven-gene operon additionally including mgsA, encoding methylglyoxal synthase, and the essential genes cca and birA, encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The bshB2 gene is co-transcribed with unknown function genes, while bshC is expressed both as part of a two-gene operon (with the upstream putative pantothenate biosynthesis gene ylbQ) and from its own promoter. All three operons are expressed at a reduced level in an spx null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox centre of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an spx null mutant.

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“…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). While three of the four proteins responsible for the conversion of pantothenate (Pan; vitamin B 5 ) to CoASH in Escherichia coli are conserved in B. anthracis (10,26), a novel type III pantothenate kinase (PanK Ba ; encoded by coaX [BA0065]) (25) catalyzes the first committed step in the biosynthetic pathway in B. anthracis.…”

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“…While three of the four proteins responsible for the conversion of pantothenate (Pan; vitamin B 5 ) to CoASH in Escherichia coli are conserved in B. anthracis (10,26), a novel type III pantothenate kinase (PanK Ba ; encoded by coaX [BA0065]) (25) catalyzes the first committed step in the biosynthetic pathway in B. anthracis. The type III pantothenate kinases (PanKs), first characterized in 2005 from Bacillus subtilis strain 168 and Helicobacter pylori (6), exhibit two particularly significant differences from the type I E. coli PanK (20); the type III enzymes are not subject to feedback inhibition by CoASH, and they do not recognize the Pan antimetabolite (alternate substrate) N-pentylpantothenamide.…”

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confidence: 99%

“…The type III pantothenate kinases (PanKs), first characterized in 2005 from Bacillus subtilis strain 168 and Helicobacter pylori (6), exhibit two particularly significant differences from the type I E. coli PanK (20); the type III enzymes are not subject to feedback inhibition by CoASH, and they do not recognize the Pan antimetabolite (alternate substrate) N-pentylpantothenamide. Bioinformatics analyses (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.…”

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The Type III Pantothenate Kinase Encoded by coaX Is Essential for Growth of Bacillus anthracis

et al. 2008

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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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Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Cited by 52 publications

References 64 publications

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx

The Type III Pantothenate Kinase Encoded by coaX Is Essential for Growth of Bacillus anthracis

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Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

Cited by 52 publications

References 64 publications

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx

The Type III Pantothenate Kinase Encoded by coaX Is Essential for Growth of Bacillus anthracis

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Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,