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.     

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.     

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.     

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.     

Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was developed to allow rapid access to a small library of pantothenate analogs diversified at the β-alanine moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Escherichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be equivalent to the benchmark N-pentylpantothenamide (N5-pan) as substrates of EcPanK, while two others, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collection of data provides insight for the future design of PanK substrates in the production of useful CoA analogues.     

The crystal structure of a novel phosphopantothenate synthetase from the hyperthermophilic archaea, Thermococcus onnurineus NA1

Pantothenate is the essential precursor of coenzyme A (CoA), a fundamental cofactor in all aspects of metabolism. In bacteria and eukaryotes, pantothenate synthetase (PS) catalyzes the last step in the pantothenate biosynthetic pathway, and pantothenate kinase (PanK) phosphorylates pantothenate for its entry into the CoA biosynthetic pathway. However, genes encoding PS and PanK have not been identified in archaeal genomes. Recently, a comparative genomic analysis and the identification and characterization of two novel archaea-specific enzymes show that archaeal pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) represent counterparts to the PS/PanK pathway in bacteria and eukaryotes. The TON1374 protein from Thermococcus onnurineus NA1 is a PPS, that shares 54% sequence identity with the first reported archaeal PPS candidate, MM2281, from Methanosarcina mazei and 91% sequence identity with TK1686, the PPS from Thermococcus kodakarensis. Here, we report the apo and ATP-complex structures of TON1374 and discuss the substrate-binding mode and reaction mechanism.     

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.     

A discovery of novel Mycobacterium tuberculosis pantothenate synthetase inhibitors based on the molecular mechanism of actinomycin D inhibition

Mycobacterium tuberculosis pantothenate synthetase is a potential anti-tuberculosis target, and a high-throughput screening system was previously developed to identify its inhibitors. Using a similar system, we screened a small library of compounds and identified actinomycin D (ActD) as a weak inhibitor of pantothenate synthetase. A new method was established to discover more effective inhibitors by determining the molecular mechanism of ActD inhibition followed by structure-based virtual screening. The molecular interaction of inhibition was determined by circular dichroism and tryptophan fluorescence quenching. The structure-based search and virtual screening were performed using the Molecular Operating Environment (MOE) program and SYBYL 7.5, respectively. Two inhibitors were identified with an IC₅₀ for pantothenate synthetase that was at least ten times better than that of ActD.     

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.     

Crystal structures of Klebsiella pneumoniae pantothenate kinase in complex with N‐substituted pantothenamides

N‐Substituted pantothenamides are derivatives of pantothenate, the precursor in the biosynthesis of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis and possess antimicrobial activity against various pathogenic bacteria. Here we solved the crystal structure of the Klebsiella pneumoniae PanK (KpPanK) in complex with N‐pentylpantothenamide (N5‐Pan) to understand the molecular basis of its antimicrobial activity. The structure reveals a polar pocket interacting with the pantothenate moiety of N5‐Pan and an aromatic pocket loosely protecting the pentyl tail, suggesting that the introduction of an aromatic ring to a new pantothenamide may enhance the compound's affinity to KpPanK. To test this idea, we synthesized N‐pyridin‐3‐ylmethylpantothenamide (Np‐Pan) and solved its co‐crystal structure with KpPanK. The structure reveals two alternat conformations of the aromatic ring of Np‐Pan bound at the aromatic pocket, providing the basis for further improvement of pantothenamide binding to KpPanK. Proteins 2013; 81:1466–1472. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.     

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.     

Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.     

Compartmentalization of Mammalian Pantothenate Kinases

The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.     

Crystal structure of a type III pantothenate kinase: insight into the mechanism of an essential coenzyme A biosynthetic enzyme universally distributed in bacteria

Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK(Tm)), solved at 2.0-A resolution. The structure of PanK(Tm) reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK(Tm) active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK -- which is a member of the "P-loop kinase"superfamily -- this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.     

Pantoate Kinase and Phosphopantothenate Synthetase, Two Novel Enzymes Necessary for CoA Biosynthesis in the Archaea

Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and β-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4′-phosphopantothenate. When we examined the cell-free extracts of the hyperthermophilic archaeon Thermococcus kodakaraensis, PanK activity could not be detected. A search for putative kinase-encoding genes widely distributed in Archaea, but not present in bacteria/eukaryotes, led to four candidate genes. Among these genes, TK2141 encoded a protein with relatively low PanK activity. However, higher levels of activity were observed when pantothenate was replaced with pantoate. V_max values were 7-fold higher toward pantoate, indicating that TK2141 encoded a novel enzyme, pantoate kinase (PoK). A search for genes with a distribution similar to TK2141 led to the identification of TK1686. The protein product catalyzed the ATP-dependent conversion of phosphopantoate and β-alanine to produce 4′-phosphopantothenate and did not exhibit PS activity, indicating that TK1686 also encoded a novel enzyme, phosphopantothenate synthetase (PPS). Although the classic PS/PanK system performs condensation with β-alanine prior to phosphorylation, the PoK/PPS system performs condensation after phosphorylation of pantoate. Gene disruption of TK2141 and TK1686 led to CoA auxotrophy, indicating that both genes are necessary for CoA biosynthesis in T. kodakaraensis. Homologs of both genes are widely distributed among the Archaea, suggesting that the PoK/PPS system represents the pathway for 4′-phosphopantothenate biosynthesis in the Archaea.Coenzyme A (CoA)² and its derivative 4′-phosphopantetheine are essential cofactors in numerous metabolic pathways, including the tricarboxylic acid cycle, the β-oxidation pathway, and fatty acid and polyketide biosynthesis pathways. Acyl-CoA derivatives are key intermediates in energy metabolism due to their high energy thioester bonds and have been identified in all three domains of life.The mechanism of CoA biosynthesis in bacteria and eukaryotes has been well examined and involves common enzymatic conversions (1–3). CoA is synthesized from pantothenate via five enzymatic reactions; pantothenate kinase (PanK), 4′-phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantothenoylcysteine decarboxylase (PPCDC), 4′- phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK). Although many animals rely on exogenous pantothenate to initiate CoA biosynthesis, microorganisms and plants can synthesize pantothenate from 2-oxoisovalerate and β-alanine. This is a three-step pathway catalyzed by ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase, and pantothenate synthetase (PS).In contrast to the wealth of knowledge on CoA biosynthesis in bacteria and eukaryotes, the corresponding pathway in the Archaea remains unclear (4). Sequence data indicate that the bacterial PPCS and PPCDC homologs and eukaryotic PPAT homologs are found on almost all of the archaeal genomes. The archaeal PPCS and PPCDC genes are fused in many cases, and the bifunctional protein from Methanocaldococcus jannaschii has been shown to exhibit both activities (5). The PPAT homolog from Pyrococcus abyssi has also been studied and confirmed to exhibit the expected PPAT activity (6). Bacterial KPHMT and ketopantoate reductase homologs can also be found, to a lesser extent, on the archaeal genomes. They are not found in the methanogens and Thermoplasmatales, and the fact that the structural similarity among archaeal enzymes is not higher than that toward enzymes from hyperthermophilic bacteria suggests that the archaeal KPHMT and ketopantoate reductase are a result of horizontal gene transfer from bacteria (4). In addition, there are candidate genes distantly related to bacterial/eukaryotic DPCK. However, PS homologs are not found in any of the archaeal genomes, and PanK homologs are found only in a few exceptional cases. Recently, Genschel and co-workers have taken a comparative genomics approach to predict the genes corresponding to the archaeal PS and PanK genes, and have also described the identification of a structurally novel PS from Methanosarcina mazei (4, 7).In this study, we describe the identification of the enzymes responsible for the conversion of pantoate to 4′-phosphopantothenate in Thermococcus kodakaraensis. The organism is a hyperthermophilic archaeon isolated from Kodakara Island, Japan (8, 9). The complete genome sequence is available (10), and gene disruption systems have been developed (11–13). To our surprise, the conversion of pantoate to 4′-phosphopantothenate in T. kodakaraensis is not brought about by the two classic enzyme reactions catalyzed by PS and PanK, but by two novel enzyme reactions; phosphorylation of pantoate (pantoate kinase) followed by the condensation of 4-phosphopantoate and β-alanine (4′-phosphopantothenate synthetase or 4-phosphopantoate:β-alanine ligase). Homologs of these two genes are distributed on almost all of the archaeal genomes, suggesting that the Archaea utilize different chemistry in the conversion from pantoate to 4′-phosphopantothenate.