Structural basis for substrate binding and the catalytic mechanism of type III pantothenate kinase

Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high Km for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the "acetate and sugar kinase/heat shock protein 70/actin" (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.     

Crystal structures of human pantothenate kinases. Insights into allosteric regulation and mutations linked to a neurodegeneration disorder

Pantothenate kinase (PanK) catalyzes the first step in CoA biosynthesis and there are three human genes that express four isoforms with highly conserved catalytic core domains. Here we report the homodimeric structures of the catalytic cores of PanK1alpha and PanK3 in complex with acetyl-CoA, a feedback inhibitor. Each monomer adopts a fold of the actin kinase superfamily and the inhibitor-bound structures explain the basis for the allosteric regulation by CoA thioesters. These structures also provide an opportunity to investigate the structural effects of the PanK2 mutations that have been implicated in neurodegeneration. Biochemical and thermodynamic analyses of the PanK3 mutant proteins corresponding to PanK2 mutations show that mutant proteins with compromised activities and/or stabilities correlate with a higher incidence of the early onset of disease.     

Structural basis for the feedback regulation of Escherichia coli pantothenate kinase by coenzyme A

Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate. CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site. The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively. Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface. The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP. Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state. The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters. The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.     

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

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.     

Probing the ligand preferences of the three types of bacterial pantothenate kinase

Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4′-phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here, a series of pantothenate analogues were synthesized using convenient synthetic methodology. The compounds were exploited as small organic probes to compare the ligand preferences of the three different types of bacterial PanK. Overall, several new inhibitors and substrates were identified for each type of PanK.     

A novel heteromeric pantothenate kinase complex in apicomplexan parasites

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.     

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.     

Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration

The PANK2 gene encodes the human pantothenate kinase 2 protein isoforms, and PANK2 mutations are linked to pantothenate kinase-associated neurodegeneration. Two PanK2 protein forms are proteolytically processed to form a mitochondrially localized, mature PanK2. Another isoform arose from a proposed initiation at a leucine codon and was not processed further. The fifth isoform was postulated to arise from an alternative splicing event and was found to encode an inactive protein. Fourteen mutant PanK2 proteins with single amino acid substitutions, associated with either early or late onset disease, were evaluated for activity. The PanK2(G521R), the most frequent mutation in pantothenate kinase-associated neurodegeneration, was devoid of activity and did not fold properly. However, nine of the mutant proteins associated with disease possessed catalytic activities that were indistinguishable from wild type, including the frequently encountered PanK2(T528M) missense mutation. PanK2 was extremely sensitive to feedback inhibition by CoA thioesters (IC50 values between 250 and 500 nM), and the regulation of the active PanK2 mutants was comparable with that of the wild-type protein. Coexpression of the PanK2(G521R) and wild-type PanK2 did not interfere with wild-type enzyme activity, arguing against a dominant negative effect of the PanK2(G521R) mutation in heterozygous patients. These data described the unique biochemical features of the PanK2 isoforms and suggested that catalytic defects may not be the sole cause for the neurodegenerative phenotype.     

Localization and regulation of mouse pantothenate kinase 2

Coenzyme A (CoA) biosynthesis is initiated by pantothenate kinase (PanK) and CoA levels are controlled through differential expression and feedback regulation of PanK isoforms. PanK2 is a mitochondrial protein in humans, but comparative genomics revealed that acquisition of a mitochondrial targeting signal was limited to primates. Human and mouse PanK2 possessed similar biochemical properties, with inhibition by acetyl-CoA and activation by palmitoylcarnitine. Mouse PanK2 localized in the cytosol, and the expression of PanK2 was higher in human brain compared to mouse brain. Differences in expression and subcellular localization should be considered in developing a mouse model for human PanK2 deficiency.     

Feedback regulation of murine pantothenate kinase 3 by coenzyme A and coenzyme A thioesters

Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1beta isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1beta and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC50 = 1 microm) than PanK1beta (IC50 = 10 microm), and PanK3 was stringently regulated by long-chain acyl-CoA (IC50 = 2 microm), whereas PanK1beta was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1beta. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.     

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.     

Pantothenate kinase regulation of the intracellular concentration of coenzyme A

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1alpha and mPanK1beta) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1alpha was most highly expressed in heart and kidney, whereas mPanK1beta mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1beta expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1beta activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1beta activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.     

Characterization of a new pantothenate kinase isoform from Helicobacter pylori

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.     

Structural characterization of a new N‐substituted pantothenamide bound to pantothenate kinases from Klebsiella pneumoniae and Staphylococcus aureus

Pantothenate kinase (PanK) is the rate‐limiting enzyme in Coenzyme A biosynthesis, catalyzing the ATP‐dependent phosphorylation of pantothenate. We solved the co‐crystal structures of PanKs from Staphylococcus aureus (SaPanK) and Klebsiella pneumonia (KpPanK) with N‐[2‐(1,3‐benzodioxol‐5‐yl)ethyl] pantothenamide (N354‐Pan). Two different N354‐Pan conformers interact with polar/nonpolar mixed residues in SaPanK and aromatic residues in KpPanK. Additionally, phosphorylated N354‐Pan is found at the closed active site of SaPanK but not at the open active site of KpPanK, suggesting an exchange of the phosphorylated product with a new N354‐Pan only in KpPanK. Together, pantothenamides conformational flexibility and binding pocket are two key considerations for selective compound design. Proteins 2014; 82:1542–1548. © 2014 Wiley Periodicals, Inc.     

Structure of serine acetyltransferase in complexes with CoA and its cysteine feedback inhibitor

Serine acetyltransferase (SAT, EC 2.3.1.30) catalyzes the CoA-dependent acetylation of the side chain hydroxyl group of l-serine to form O-acetylserine, as the first step of a two-step biosynthetic pathway in bacteria and plants leading to the formation of l-cysteine. This reaction represents a key metabolic point of regulation for the cysteine biosynthetic pathway due to its feedback inhibition by cysteine. We have determined the X-ray crystal structure of Haemophilus influenzae SAT in complexes with CoA and its cysteine feedback inhibitor. The enzyme is a 175 kDa hexamer displaying the characteristic left-handed parallel beta-helix (LbetaH) structural domain of the hexapeptide acyltransferase superfamily of enzymes. Cysteine is bound in a crevice between adjacent LbetaH domains and underneath a loop excluded from the coiled LbetaH. The proximity of its thiol group to the thiol group of CoA derived from superimposed models of the cysteine and CoA complexes confirms that cysteine is bound at the active site. Analysis of the contacts of SAT with cysteine and CoA and the conformational differences that distinguish these complexes provides a structural basis for cysteine feedback inhibition, which invokes competition between cysteine and serine binding and a cysteine-induced conformational change of the C-terminal segment of the enzyme that excludes binding of the cofactor.     

Identification,purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mm. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.     

Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts.

The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.     

Structure-activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors

A set of novel pantothenamide-type analogues of the known Staphylococcus aureus pantothenate kinase (SaPanK) inhibitors, N-pentyl, and N-heptylpantothenamide, was synthesized in three series. The first series of analogues (1-3) were designed as molecular probes of the PanK binding site to elucidate important structure-activity relationships (SAR). The second series of analogues (4-16) were designed using structural information obtained from the Escherichia coli PanK (EcPanK) structure by targeting the pantothenate binding site and the adjacent phenylalanine-lined lipophilic pocket. Insight into the antimicrobial effect of N-pentylpantothenamide (N5-Pan) through its conversion to the antimetabolite ethyldethia-CoA and further incorporation into an inactive acyl carrier protein analogue drove the development of the third series of analogues (17-25) to enhance this effect using substrate-like substitutions. Each of the analogues was screened for enzyme inhibition activity against a panel of pantothenate kinases consisting of EcPanK, Aspergillus nidulans (AnPanK), SaPanK, and the murine isoform (MmPanK1alpha). Series 1 demonstrated only modest inhibitory activity, but did reveal some important SAR findings including stereospecific binding. Series 2 demonstrated a much higher inhibition rate for the entire series and significant inhibition was seen with analogues containing alkyl substituents. Series 3 demonstrated the most preferential inhibition profile, with the highest inhibitory activity against the SaPanK and MmPanK1alpha. The MmPanK1alpha protein was inhibited by a broad spectrum of the compounds, whereas the E. coli enzyme showed greater selectivity. The overall activity data from these analogues suggest a complex and non-enzyme specific SAR for pantothenamide substrate/inhibitors of the different PanK enzymes.