Reactions Of Central Metabolism Research Articles

The alpha-ketoacid dehydrogenase complexes of Drosophila melanogaster.

The conserved family of alpha-ketoacid dehydrogenase complexes (AKDHCs) catalyze essential reactions in central metabolism and their dysregulation is implicated in several human diseases. Drosophila melanogaster provides an excellent model system to study the genetics and functions of these complexes. However, a systematic account of Drosophila AKDHCs and their composition has been lacking. Here, I identify and classify the genes encoding all Drosophila AKDHC subunits, update their functional annotations and integrate this work into the FlyBase database.

Cellular localization of the hybrid pyruvate/2-oxoglutarate dehydrogenase complex in the actinobacterium Corynebacterium glutamicum.

For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.

Production of selenium nanoparticles occurs through an interconnected pathway of sulphur metabolism and oxidative stress response in Pseudomonas putida KT2440.

The soil bacterium Pseudomonas putida KT2440 has been shown to produce selenium nanoparticles aerobically from selenite; however, the molecular actors involved in this process are unknown. Here, through a combination of genetic and analytical techniques, we report the first insights into selenite metabolism in this bacterium. Our results suggest that the reduction of selenite occurs through an interconnected metabolic network involving central metabolic reactions, sulphur metabolism, and the response to oxidative stress. Genes such as sucA, D2HGDH and PP_3148 revealed that the 2-ketoglutarate and glutamate metabolism is important to convert selenite into selenium. On the other hand, mutations affecting the activity of the sulphite reductase decreased the bacteria's ability to transform selenite. Other genes related to sulphur metabolism (ssuEF, sfnCE, sqrR, sqr and pdo2) and stress response (gqr, lsfA, ahpCF and sadI) were also identified as involved in selenite transformation. Interestingly, suppression of genes sqrR, sqr and pdo2 resulted in the production of selenium nanoparticles at a higher rate than the wild-type strain, which is of biotechnological interest. The data provided in this study brings us closer to understanding the metabolism of selenium in bacteria and offers new targets for the development of biotechnological tools for the production of selenium nanoparticles.

2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo

Pyridoxal 5′-phosphate (PLP) is an essential cofactor for a class of enzymes that catalyze diverse reactions in central metabolism. The catalytic mechanism of some PLP-dependent enzymes involves the generation of reactive enamine intermediates like 2-aminoacrylate (2AA). 2AA can covalently modify PLP in the active site of some PLP-dependent enzymes and subsequently inactivate the enzyme through the formation of a PLP–pyruvate adduct. In the absence of the enamine/imine deaminase RidA, Salmonella enterica experiences 2AA-mediated metabolic stress. Surprisingly, PLP-dependent enzymes that generate endogenous 2AA appear to be immune to its attack, while other PLP-dependent enzymes accumulate damage in the presence of 2AA stress; however, structural determinants of 2AA sensitivity are unclear. In this study, we refined a molecular method to query proteins from diverse systems for their sensitivity to 2AA in vivo. This method was then used to examine active site residues of Alr, a 2AA-sensitive PLP-dependent enzyme, that affect its sensitivity to 2AA in vivo. Unexpectedly, our data also showed that a low level of 2AA stress can persist even in the presence of a functional RidA. In summary, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the structural determinants influencing enzyme susceptibility to damage by free 2AA.

Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

The MpsAB Bicarbonate Transporter Is Superior to Carbonic Anhydrase in Biofilm-Forming Bacteria with Limited CO2 Diffusion.

ABSTRACTCO2 and bicarbonate are required for carboxylation reactions, which are essential in most bacteria. To provide the cells with sufficient CO2, there exist two dissolved inorganic carbon supply (DICS) systems: the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA). Recently, it has been shown that MpsAB is a bicarbonate transporter that is present not only in photo- and autotrophic bacteria, but also in a diverse range of nonautotrophic microorganisms. Since the two systems rarely coexist in a species but are interchangeable, we investigated what advantages the one system might have over the other. Using the genus Staphylococcus as a model, we deleted the CA gene can in Staphylococcus carnosus and mpsABC genes in Staphylococcus aureus. Deletion of the respective gene in one or the other species led to growth inhibition that could only be reversed by CO2 supplementation. While the S. carnosus Δcan mutant could be fully complemented with mpsABC, the S. aureus ΔmpsABC mutant was only partially complemented by can, suggesting that MpsAB outperforms CA. Interestingly, we provide evidence that mucus biofilm formation such as that involving polysaccharide intercellular adhesin (PIA) impedes the diffusion of CO2 into cells, making MpsAB more advantageous in biofilm-producing strains or species. Coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. In conclusion, the distribution of MpsAB or CA in bacteria does not appear to be random as expression of bicarbonate transporters provides an advantage where diffusion of CO2 is impeded.IMPORTANCE CO2 and bicarbonate are required for carboxylation reactions in central metabolism and biosynthesis of small molecules in all bacteria. This is achieved by two different systems for dissolved inorganic carbon supply (DICS): these are the membrane potential-generating system (MpsAB) and the carbonic anhydrase (CA), but both rarely coexist in a given species. Here, we compared both systems and demonstrate that the distribution of MpsAB and/or CA within the phylum Firmicutes is apparently not random. The bicarbonate transporter MpsAB has an advantage in species where CO2 diffusion is hampered—for instance, in mucus- and biofilm-forming bacteria. However, coexpression of MpsAB and CA does not confer any growth benefits, even under stress conditions. Given the clinical relevance of Staphylococcus in the medical environment, such findings contribute to the understanding of bacterial metabolism and thus are crucial for exploration of potential targets for antimicrobials. The knowledge gained here as exemplified by staphylococcal species could be extended to other pathogenic bacteria.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers ΔG⧧ for kcat/Km for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose 6-phosphate isomerase are reduced by 11–13 kcal/mol by interactions between the protein and the substrate phosphodianion. Between 4 and 6 kcal/mol of this dianion binding energy is expressed at the transition state for phosphite dianion activation of the respective enzyme-catalyzed reactions of truncated substrates d-xylonate or d-xylose. These and earlier results from studies on β-phosphoglucomutase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase define a cluster of six enzymes that catalyze reactions in glycolysis or of glycolytic intermediates, and which utilize substrate dianion binding energy for enzyme activation. Dianion-driven conformational changes, which convert flexible open proteins to tight protein cages for the phosphorylated substrate, have been thoroughly documented for five of these six enzymes. The clustering of metabolic enzymes which couple phosphodianion-driven conformational changes to enzyme activation suggests that this catalytic motif has been widely propagated in the proteome.

Enzyme activities predicted by metabolite concentrations and solvent capacity in the cell.

Experimental measurements or computational model predictions of the post-translational regulation of enzymes needed in a metabolic pathway is a difficult problem. Consequently, regulation is mostly known only for well-studied reactions of central metabolism in various model organisms. In this study, we use two approaches to predict enzyme regulation policies and investigate the hypothesis that regulation is driven by the need to maintain the solvent capacity in the cell. The first predictive method uses a statistical thermodynamics and metabolic control theory framework while the second method is performed using a hybrid optimization–reinforcement learning approach. Efficient regulation schemes were learned from experimental data that either agree with theoretical calculations or result in a higher cell fitness using maximum useful work as a metric. As previously hypothesized, regulation is herein shown to control the concentrations of both immediate and downstream product concentrations at physiological levels. Model predictions provide the following two novel general principles: (1) the regulation itself causes the reactions to be much further from equilibrium instead of the common assumption that highly non-equilibrium reactions are the targets for regulation; and (2) the minimal regulation needed to maintain metabolite levels at physiological concentrations maximizes the free energy dissipation rate instead of preserving a specific energy charge. The resulting energy dissipation rate is an emergent property of regulation which may be represented by a high value of the adenylate energy charge. In addition, the predictions demonstrate that the amount of regulation needed can be minimized if it is applied at the beginning or branch point of a pathway, in agreement with common notions. The approach is demonstrated for three pathways in the central metabolism of E. coli (gluconeogenesis, glycolysis-tricarboxylic acid (TCA) and pentose phosphate-TCA) that each require different regulation schemes. It is shown quantitatively that hexokinase, glucose 6-phosphate dehydrogenase and glyceraldehyde phosphate dehydrogenase, all branch points of pathways, play the largest roles in regulating central metabolism.

Leaf Energy Balance Requires Mitochondrial Respiration and Export of Chloroplast NADPH in the Light.

Key aspects of leaf mitochondrial metabolism in the light remain unresolved. For example, there is debate about the relative importance of exporting reducing equivalents from mitochondria for the peroxisomal steps of photorespiration versus oxidation of NADH to generate ATP by oxidative phosphorylation. Here, we address this and explore energetic coupling between organelles in the light using a diel flux balance analysis model. The model included more than 600 reactions of central metabolism with full stoichiometric accounting of energy production and consumption. Different scenarios of energy availability (light intensity) and demand (source leaf versus a growing leaf) were considered, and the model was constrained by the nonlinear relationship between light and CO2 assimilation rate. The analysis demonstrated that the chloroplast can theoretically generate sufficient ATP to satisfy the energy requirements of the rest of the cell in addition to its own. However, this requires unrealistic high light use efficiency and, in practice, the availability of chloroplast-derived ATP is limited by chloroplast energy dissipation systems, such as nonphotochemical quenching, and the capacity of the chloroplast ATP export shuttles. Given these limitations, substantial mitochondrial ATP synthesis is required to fulfill cytosolic ATP requirements, with only minimal, or zero, export of mitochondrial reducing equivalents. The analysis also revealed the importance of exporting reducing equivalents from chloroplasts to sustain photorespiration. Hence, the chloroplast malate valve and triose phosphate-3-phosphoglycerate shuttle are predicted to have important metabolic roles, in addition to their more commonly discussed contribution to the avoidance of photooxidative stress.

Quantum chemistry reveals thermodynamic principles of redox biochemistry.

Thermodynamics dictates the structure and function of metabolism. Redox reactions drive cellular energy and material flow. Hence, accurately quantifying the thermodynamics of redox reactions should reveal design principles that shape cellular metabolism. However, only few redox potentials have been measured, and mostly with inconsistent experimental setups. Here, we develop a quantum chemistry approach to calculate redox potentials of biochemical reactions and demonstrate our method predicts experimentally measured potentials with unparalleled accuracy. We then calculate the potentials of all redox pairs that can be generated from biochemically relevant compounds and highlight fundamental trends in redox biochemistry. We further address the question of why NAD/NADP are used as primary electron carriers, demonstrating how their physiological potential range fits the reactions of central metabolism and minimizes the concentration of reactive carbonyls. The use of quantum chemistry can revolutionize our understanding of biochemical phenomena by enabling fast and accurate calculation of thermodynamic values.

Phosphoglycerate kinase acts as a futile cycle at high temperature.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

An analysis of the distribution of key metabolic fluxes in Chlamydomonas reinhardtii cells under the conditions of a sulfur deficit

A stoichiometric model that combines the reactions of central metabolism and the electron transport processes of respiration and primary photosynthetic reactions in a plant cell is considered. The central metabolic reactions, including glycolysis, the Calvin cycle, and the Krebs cycle, are associated with electrontransport processes via the NAD(P)H redox equivalents. The model is defined by algebraic equations and includes rules that enable the description of the change in the direction of metabolism dependent on the availability of different carbon sources. Experimental data on the changes of the levels of individual cellular metabolites in the alga Chlamydomonas reinhardtii under the conditions of sulfur deprivation were used to verify the model. The model allowed the generalization of the existing concepts of the changes in the direction of metabolic fluxes under the conditions of sulfur starvation.

Interaction of 2-oxoglutarate dehydrogenase OdhA with its inhibitor OdhI in Corynebacterium glutamicum: Mutants and a model

Pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. In Corynebacterium glutamicum and related bacteria like Mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein OdhA catalyzing the conversion of oxoglutarate to succinyl-coenzyme A. This activity is inhibited by the forkhead-associated (FHA) domain of the small autoinhibitory protein OdhI. Here we used a biological screen which enabled us to isolate suppressor mutants that are influenced in OdhA–OdhI interaction. Five mutants carrying an OdhI mutation were isolated and one with an OdhA mutation. The OdhA mutein OdhA-C704E and three additional C704 variants were constructed. They exhibited unaltered or even slightly enhanced OdhA activity but showed reduced inhibition and interaction with OdhI. The FHA domain of OdhI was crystallized and its structure found in full agreement with previously determined NMR structures. Based on further structural studies, OdhA-OdhI crosslinking experiments, and modeling we discuss the experimental data generated on OdhA–OdhI interaction, with the latter protein representing a rare example of an FHA domain also recognizing a non-phosphorylated interaction partner.

Structural Basis for Universal Corrinoid Recognition by the Cobalamin Transport Protein Haptocorrin

Cobalamin (Cbl; vitamin B12) is an essential micronutrient synthesized only by bacteria. Mammals have developed a sophisticated uptake system to capture the vitamin from the diet. Cbl transport is mediated by three transport proteins: transcobalamin, intrinsic factor, and haptocorrin (HC). All three proteins have a similar overall structure but a different selectivity for corrinoids. Here, we present the crystal structures of human HC in complex with cyanocobalamin and cobinamide at 2.35 and 3.0 Å resolution, respectively. The structures reveal that many of the interactions with the corrin ring are conserved among the human Cbl transporters. However, the non-conserved residues Asn-120, Arg-357, and Asn-373 form distinct interactions allowing for stabilization of corrinoids other than Cbl. A central binding motif forms interactions with the e- and f-side chains of the corrin ring and is conserved in corrinoid-binding proteins of other species. In addition, the α- and β-domains of HC form several unique interdomain contacts and have a higher shape complementarity than those of intrinsic factor and transcobalamin. The stabilization of ligands by all of these interactions is reflected in higher melting temperatures of the protein-ligand complexes. Our structural analysis offers fundamental insights into the unique binding behavior of HC and completes the picture of Cbl interaction with its three transport proteins.

Evaluation of control mechanisms for Saccharomyces cerevisiae central metabolic reactions using metabolome data of eight single-gene deletion mutants

We performed metabolome and metabolite-metabolite correlation analyses for eight single-gene deletion mutants of Saccharomyces cerevisiae to evaluate the physiology of glucose metabolism. The irreversible enzyme reactions can become bottlenecks when intracellular metabolism is perturbed by direct interference from the central metabolic pathway by gene deletions or by a deletion of transcriptional regulator. Metabolome data reveal that transcriptional factor, gcr2, regulates the reaction that converts 3-phosphoglycerate into phosphoenolpyruvate. Metabolome data also suggest that the reaction catalyzed by pyruvate kinase makes one of the rate-limiting reactions throughout the glycolytic pathway.

A Comparative Analysis of the Molecular Characteristics of the Arabidopsis CoA Pyrophosphohydrolases AtNUDX11, 15, and 15a

Coenzyme A (CoA) is an essential, ubiquitous cofactor in all biological systems, where it acts as the major acyl group carrier in various central metabolic reactions. Although much is known about CoA biosynthesis, it is unclear how the CoA pool is regulated the various cellular compartments. It has been found that the nucleoside diphosphates linked to some moiety X (Nudix) hydrolases, AtNUDX11 and 15, have pyrophosphohydrolase activity toward CoA and its derivatives. In this study we identified two alternatively spliced variants, AtNUDX15 and 15a, produced from the AtNUDX15 gene, and carried out comparative studies of the gene regulation, the kinetic parameters, and the intracellular localization of AtNUDX11, 15, and 15a. The present findings indicate that AtNUDX11 and AtNUDX15(a) function in the hydrolysis of malonyl-CoA in cytosol and succinyl-CoA in the mitochondria, respectively, suggesting their impact not only on CoA biosynthesis but also on various CoA-related pathways such as the TCA cycle.

The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as C. glutamicum and M. tuberculosis.

Invariability of central metabolic flux distribution in Shewanella oneidensis MR‐1 under environmental or genetic perturbations

An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time approximately 3 h), salt stressed minimal medium (doubling time approximately 6 h), and minimal medium with amino acid supplementation (doubling time approximately 1.5 h). (13)C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, 10 transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.

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.