Coenzyme A Biosynthesis Research Articles

Antiplasmodial Mode of Action of Pantothenamides: Pantothenate Kinase Serves as a Metabolic Activator Not as a Target.

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural β-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites and PanAm phosphorylation by PfPanK present in parasite lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the β-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.

Discovery and Development of Allosteric Pantothenate Kinase Activators

The central position of coenzyme A (CoA) in intermediary metabolism and its role in human neurodegenerative disease, led us to develop chemical probes targeting pantothenate kinase (PANK), the regulatory enzyme that controls CoA biosynthesis. There are four mammalian PANK isoforms that exhibit tissue‐specific expression and differ in their sensitivity to feedback inhibition by CoA and its thioesters. A high‐throughput screen and further medicinal chemistry efforts identified a novel class of orally bioavailable PanK modulators called pantazines. Optimization of the series based on lipophilic ligand efficiency (LipE) culminated in PZ‐2891, a drug‐like molecule that binds to PANK3 with nM affinity. Enzyme kinetics, biophysical studies, and X‐ray crystallography demonstrate that PZ‐2891 binds to the pantothenate substrate site of the PANK3•ATP•Mg2+ complex and extends across the dimer interface to form an induced fit, high‐affinity quaternary complex. PANK3 is a highly cooperative dimer and both protomer active sites simultaneously switch between the closed, active conformation that binds ATP, and the open, inactive conformation that binds acetyl‐CoA. PZ‐2891 inactivates the protomer to which it is bound and locks the opposite protomer in an active conformation that is refractory to feedback regulation by acetyl‐CoA. This allosteric property means that PZ‐2891 is a pantothenate‐dependent activator of CoA biosynthesis in cultured cells, and substantially increases intracellular CoA levels as a function of the media pantothenate supplement. Administration of PZ‐2891 to animals by oral gavage recapitulated the pantothenate‐dependent modulation of CoA levels in liver. PZ‐2891 dose‐response experiments also demonstrated the elevation of brain CoA illustrating the ability of PZ‐2891 to cross the blood brain barrier. PZ‐2891 and its inactive analogs are ready to be deployed as a toolkit to manipulate CoA levels in cultured cells and animals to analyze the role of CoA concentration in the control of intermediary metabolism. These nanomolar PANK allosteric effectors are being further developed as preclinical leads to validate PANK as a target to treat neurologic and metabolic disorders.Support or Funding InformationNIH, ALSAC/St. Jude

A novel small RNA CoaR regulates coenzyme A biosynthesis and tolerance of Synechocystis sp. PCC6803 to 1-butanol possibly via promoter-directed transcriptional silencing

BackgroundMicrobial small RNAs (sRNAs) have been proposed as valuable regulatory elements for optimizing cellular metabolism for industrial purposes. However, little information is currently available on functional relevance of sRNAs to biofuels tolerance in cyanobacteria.ResultsHere, we described the identification and functional characterization of a novel 124 nt sRNA Ncl1460 involved in tolerance to biofuel 1-butanol in Synechocystis sp. PCC 6803. The expression of Ncl1460 was verified by blotting assay and its length was determined through 3′ RACE. Further analysis showed that Ncl1460 was a negative regulator of slr0847 (coaD) and slr0848 operon responsible for coenzyme A (CoA) synthesis possibly via promoter-directed transcriptional silencing mechanisms which has been widely discovered in eukaryote; thus Ncl1460 was designated as CoaR (CoA Biosynthesis Regulatory sRNA). The possible interaction between CoaR and target genes was suggested by CoA quantification and green fluorescent protein assays. Finally, a quantitative proteomics analysis showed that CoaR regulated tolerance to 1-butanol possibly by down-regulating CoA biosynthesis, resulting in a decrease of fatty acid metabolism and energy metabolism.ConclusionsAs the first reported sRNA involved CoA synthesis and 1-butanol tolerance in cyanobacteria, this study provides not only novel insights in regulating mechanisms of essential pathways in cyanobacteria, but also valuable target for biofuels tolerance and productivity modifications.

Down-regulation of coasy, the gene associated with NBIA-VI, reduces Bmp signaling, perturbs dorso-ventral patterning and alters neuronal development in zebrafish.

Mutations in Pantothenate kinase 2 and Coenzyme A (CoA) synthase (COASY), genes involved in CoA biosynthesis, are associated with rare neurodegenerative disorders with brain iron accumulation. We showed that zebrafish pank2 gene plays an essential role in brain and vasculature development. Now we extended our study to coasy. The gene has high level of sequence identity with the human ortholog and is ubiquitously expressed from the earliest stages of development. The abrogation of its expression led to strong reduction of CoA content, high lethality and a phenotype resembling to that of dorsalized mutants. Lower doses of morpholino resulted in a milder phenotype, with evident perturbation in neurogenesis and formation of vascular arborization; the dorso-ventral patterning was severely affected, the expression of bone morphogenetic protein (Bmp) receptors and activity were decreased, while cell death increased. These features specifically correlated with the block in CoA biosynthesis and were rescued by the addition of CoA to fish water and the overexpression of the human wild-type, but not mutant gene. These results confirm the absolute requirement for adequate levels of CoA for proper neural and vascular development in zebrafish and point to the Bmp pathway as a possible molecular connection underlining the observed phenotype.

Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

Triazole Substitution of a Labile Amide Bond Stabilizes Pantothenamides and Improves Their Antiplasmodial Potency.

The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC50s) of ∼50 nM, and three others have submicromolar IC50s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [14C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase.

Regulation of Coenzyme A Biosynthesis in the Hyperthermophilic Bacterium Thermotoga maritima.

Regulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. maritima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated Tm-KPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase (Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 μM CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated by feedback inhibition targeting PanK, although Tm-PanK is a type III enzyme. Bacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase (PanK). The hyperthermophilic bacterium Thermotoga maritima harbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found that Tm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated in T. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.

Identifying vulnerable steps in the coenzyme A biosynthesis pathway of M. tuberculosis

Enzymes in the pantothenate and coenzyme A (CoA) biosynthesis pathways have attracted considerable interest as potential targets for the development of drugs against a number of human pathogens, including M. tuberculosis. While potent inhibitors have been developed against M. tuberculosis pantothenate synthase and pantothenate kinase, these have failed to translate into compounds with significant whole‐cell activity. These findings illustrate the challenges inherent in target‐led approaches to tuberculosis drug discovery, which is confounded by issues of permeability, metabolism and efflux, by a lack of understanding of target vulnerability.To identify vulnerable steps in the pantothenate and CoA biosynthesis pathway in M. tuberculosis, a set of anhydrotetracycline (ATc)‐regulated mutants of M. tuberculosis was constructed using a transcriptional silencing system. The mutants were used to assess the impact of depletion of specific targets in the pantothenate and CoA pathway on growth and survival of the organism. Mutants with inducer‐dependent growth phenotypes were obtained for panB, panC, coaBC, coaD and coaE whereas those in panB, panE and panK were refractory to ATc‐regulated growth. While transcriptional silencing of panB, panC or coaE was bacteriostatic, coaBC silencing was apparently bactericidal in vitro, as determined by CFU enumeration. CoaBC was likewise shown to be required for growth and persistence of M. tuberculosis in vivo based on enumeration of bacillary loads in the lungs of mice in which coaBC was silenced immediately post‐infection, during acute infection, or during chronic infection. However, by taking advantage of the fact that M. tuberculosis is capable of CoaBC bypass via CoA salvage, coaBC silencing was shown to result in a transient, ‘non‐growing but metabolically active’ state from which non‐culturable bacilli can be rescued by uptake and assimilation of pantethine. These findings highlight the limitations of colony‐forming ability as a proxy for viability in M. tuberculosis. In further studies, the impact of silencing of these genes is being investigated by analysing changes in the metabolomic profile of M. tuberculosis with the aim of identifying similarities and differences in the metabolic response to target depletion at different steps of the pathway.Support or Funding InformationThis work was supported by grants from the TB Drug Accelerator program of the Bill and Melinda Gates Foundation (to C.E.B. III, D.S., K.R. and V.M.), the South African Medical Research Council, the National Research Foundation of South Africa and an International Research Scholar's grant from the HHMI (to V.M.) and, in part, by the Intramural Research Program of NIAID (H.B. and C.E.B. III).

Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2-oxopantoate provides structural insights into feedback regulation.

Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2-oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2-oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea.

Vitamin B5 and N-Acetylcysteine in Nonalcoholic Steatohepatitis: A Preclinical Study in a Dietary Mouse Model.

Nonalcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease and second indication for liver transplantation in the Western world. Effective therapy is still not available. Previously we showed a critical role for caspase-2 in the pathogenesis of nonalcoholic steatohepatitis (NASH), the potentially progressive form of NAFLD. An imbalance between free coenzyme A (CoA) and acyl-CoA ratio is known to induce caspase-2 activation. We aimed to evaluate CoA metabolism and the effects of supplementation with CoA precursors, pantothenate and cysteine, in mouse models of NASH. CoA metabolism was evaluated in methionine-choline deficient (MCD) and Western diet mouse models of NASH. MCD diet-fed mice were treated with pantothenate and N-acetylcysteine or placebo to determine effects on NASH. Liver free CoA content was reduced, pantothenate kinase (PANK), the rate-limiting enzyme in the CoA biosynthesis pathway, was down-regulated, and CoA degrading enzymes were increased in mice with NASH. Decreased hepatic free CoA content was associated with increased caspase-2 activity and correlated with worse liver cell apoptosis, inflammation, and fibrosis. Treatment with pantothenate and N-acetylcysteine did not inhibit caspase-2 activation, improve NASH, normalize PANK expression, or restore free CoA levels in MCD diet-fed mice. In mice with NASH, hepatic CoA metabolism is impaired, leading to decreased free CoA content, activation of caspase-2, and increased liver cell apoptosis. Dietary supplementation with CoA precursors did not restore CoA levels or improve NASH, suggesting that alternative approaches are necessary to normalize free CoA during NASH.

Coenzyme A biosynthetic machinery in mammalian cells.

CoA (coenzyme A) is an essential cofactor in all living organisms. CoA and its thioester derivatives [acetyl-CoA, malonyl-CoA, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) etc.] participate in diverse anabolic and catabolic pathways, allosteric regulatory interactions and the regulation of gene expression. The biosynthesis of CoA requires pantothenic acid, cysteine and ATP, and involves five enzymatic steps that are highly conserved from prokaryotes to eukaryotes. The intracellular levels of CoA and its derivatives change in response to extracellular stimuli, stresses and metabolites, and in human pathologies, such as cancer, metabolic disorders and neurodegeneration. In the present mini-review, we describe the current understanding of the CoA biosynthetic pathway, provide a detailed overview on expression and subcellular localization of enzymes implicated in CoA biosynthesis, their regulation and the potential to form multi-enzyme complexes for efficient and highly co-ordinated biosynthetic process.

Recent advances in targeting coenzyme A biosynthesis and utilization for antimicrobial drug development.

The biosynthesis and utilization of CoA (coenzyme A), the ubiquitous and essential acyl carrier in all organisms, have long been regarded as excellent targets for the development of new antimicrobial drugs. Moreover, bioinformatics and biochemical studies have highlighted significant differences between several of the bacterial enzyme targets and their human counterparts, indicating that selective inhibition of the former should be possible. Over the past decade, a large amount of structural and mechanistic data has been gathered on CoA metabolism and the CoA biosynthetic enzymes, and this has facilitated the discovery and development of several promising candidate antimicrobial agents. These compounds include both target-specific inhibitors, as well as CoA antimetabolite precursors that can reduce CoA levels and interfere with processes that are dependent on this cofactor. In the present mini-review we provide an overview of the most recent of these studies that, taken together, have also provided chemical validation of CoA biosynthesis and utilization as viable targets for antimicrobial drug development.

Defective pantothenate metabolism and neurodegeneration.

Inborn errors of CoA (coenzyme A) biosynthesis lead to neurodegenerative disorders in humans. PKAN (pantothenate kinase-associated neurodegeneration) manifests with damage to brain, retina and testis and is caused by mutations in PANK2, the gene encoding the mitochondrial form of pantothenate kinase, a key regulatory enzyme in CoA synthesis. Further attention has been focused on this pathway by the recent discovery that mutations in the gene encoding CoA synthase lead to a similar neurodegenerative disorder, raising the spectre of a common mechanism of pathogenesis. How do defects in CoA production result in neurodegeneration? Why are certain tissues and cell types selectively vulnerable? And what is the underlying neurodegenerative process? Answers to some of these questions have come from animal models of disease, including flies and mice, as well as directly from humans. The damaged tissue types share key features that are likely to contribute to their selective vulnerability. These include the presence of a blood-tissue barrier, the milieu with respect to oxidative stress, tissue metabolic demand, relative expression of genes encoding similar proteins in these tissues and cell membrane composition. Substantial progress in understanding these important neurometabolic disorders has been made since the first gene discovery more than a decade ago. With rational therapeutics now in development for PKAN, we foresee prevention of neurodegeneration and hope for neuroregeneration or neuro-rescue.

Coenzyme A and its derivatives: renaissance of a textbook classic.

In 1945, Fritz Lipmann discovered a heat-stable cofactor required for many enzyme-catalysed acetylation reactions. He later determined the structure for this acetylation coenzyme, or coenzyme A (CoA), an achievement for which he was awarded the Nobel Prize in 1953. CoA is now firmly embedded in the literature, and in students' minds, as an acyl carrier in metabolic reactions. However, recent research has revealed diverse and important roles for CoA above and beyond intermediary metabolism. As well as participating in direct post-translational regulation of metabolic pathways by protein acetylation, CoA modulates the epigenome via acetylation of histones. The organization of CoA biosynthetic enzymes into multiprotein complexes with different partners also points to close linkages between the CoA pool and multiple signalling pathways. Dysregulation of CoA biosynthesis or CoA thioester homoeostasis is associated with various human pathologies and, although the biochemistry of CoA biosynthesis is highly conserved, there are significant sequence and structural differences between microbial and human biosynthetic enzymes. Therefore the CoA biosynthetic pathway is an attractive target for drug discovery. The purpose of the Coenzyme A and Its Derivatives in Cellular Metabolism and Disease Biochemical Society Focused Meeting was to bring together researchers from around the world to discuss the most recent advances on the influence of CoA, its biosynthetic enzymes and its thioesters in cellular metabolism and diseases and to discuss challenges and opportunities for the future.

Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes

NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.

Methods for measuring CoA and CoA derivatives in biological samples.

CoA (coenzyme A) is a ubiquitous and essential cofactor that acts as an acyl group carrier in biochemical reactions. Apart from participating in numerous metabolic pathways as substrates and intermediates, CoA and a number of its thioester derivatives, such as acetyl-CoA, can also directly regulate the activity of proteins by allosteric mechanisms and by affecting protein acetylation reactions. Cellular levels of CoA and CoA thioesters change under various physiological and pathological conditions. Defective CoA biosynthesis is implicated in NBIA (neurodegeneration with brain iron accumulation). However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Accurate and reliable assays for measuring CoA species in biological samples are essential for studying the roles of CoA and CoA derivatives in health and disease. The present mini-review discusses methods that are commonly used to measure CoA species in biological samples.

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.

Salvage of Coenzyme A breakdown products by the STM4195 gene product in Salmonella enterica (LB276)

Coenzyme A (CoA) is an essential cofactor required in all living organisms, where it functions primarily as an acyl‐group carrier and activator of carbonyl groups. The biosynthetic pathway generating CoA from pantothenic acid is universally conserved in prokaryotes and eukaryotes. Pantothenate is generated in a pathway that culminates in the condensation of pantoate and β‐alanine. Animals are unable to synthesize pantothenate and must salvage it, or other metabolic intermediates, to generate CoA. In Salmonella enterica, intermediates up to and including pantothenate can be fed exogenously to support CoA biosynthesis. Previous studies have shown that intermediates in CoA biosynthesis beyond pantothenate are not readily salvaged by S. enterica. The work here found that a degradation product of Coenzyme A, derived from boiling CoA, can bypass a genetic block in the final step of pantothenate biosynthesis. The data also showed that the STM4195 gene product is required to salvage this unknown metabolite, and also transports pantothenate precursors, ketopantoate and pantoate. STM4195 is a member of the sodium bile acid symporter family (SBF) of proteins. HPLC separation of a preparation of boiled CoA defined a peak with biological activity, and mass spectrometry and NMR approaches are being used to identify the compound. The results herein provide the first evidence for an active mechanism to transport ketopantoate and pantoate, and further demonstrate a role for STM4195 in the salvage of a unique CoA breakdown product.Grant Funding Source: Supported by NIH GM095837

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.

Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.