Coenzyme A: to make it or uptake it?

The consensus has been that intracellular coenzyme A (CoA) is obtained exclusively by de novo biosynthesis via a universal, conserved five-step pathway in the cell cytosol. However, old and new evidence suggest that cells (and some microorganisms) have several strategies to obtain CoA, with 4'-phosphopantetheine (P-PantSH; the fourth intermediate in the canonical CoA biosynthetic pathway) serving as a 'nexus' metabolite.

PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Coenzyme biosynthesis in response to precursor availability reveals incorporation of ß-alanine from pantothenate in prototrophic bacteria.

Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon sources and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and ß-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for ß-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets.

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

In Vitro Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.

Regulation of the CoA Biosynthetic Complex Assembly in Mammalian Cells.

Coenzyme A (CoA) is an essential cofactor present in all living cells. Under physiological conditions, CoA mainly functions to generate metabolically active CoA thioesters, which are indispensable for cellular metabolism, the regulation of gene expression, and the biosynthesis of neurotransmitters. When cells are exposed to oxidative or metabolic stress, CoA acts as an important cellular antioxidant that protects protein thiols from overoxidation, and this function is mediated by protein CoAlation. CoA and its derivatives are strictly maintained at levels controlled by nutrients, hormones, metabolites, and cellular stresses. Dysregulation of their biosynthesis and homeostasis has deleterious consequences and has been noted in a range of pathological conditions, including cancer, diabetes, Reye's syndrome, cardiac hypertrophy, and neurodegeneration. The biochemistry of CoA biosynthesis, which involves five enzymatic steps, has been extensively studied. However, the existence of a CoA biosynthetic complex and the mode of its regulation in mammalian cells are unknown. In this study, we report the assembly of all five enzymes that drive CoA biosynthesis, in HEK293/Pank1ß and A549 cells, using the in situ proximity ligation assay. Furthermore, we show that the association of CoA biosynthetic enzymes is strongly upregulated in response to serum starvation and oxidative stress, whereas insulin and growth factor signaling downregulate their assembly.

The yeast pantothenate kinase Cab1 is a master regulator of sterol metabolism and of susceptibility to ergosterol biosynthesis inhibitors.

In fungi, ergosterol is an essential component of the plasma membrane. Its biosynthesis from acetyl-CoA is the primary target of the most commonly used antifungal drugs. Here, we show that the pantothenate kinase Cab1p, which catalyzes the first step in the metabolism of pantothenic acid for CoA biosynthesis in budding yeast (Saccharomyces cerevisiae), significantly regulates the levels of sterol intermediates and the activities of ergosterol biosynthesis-targeting antifungals. Using genetic and pharmacological analyses, we show that altered pantothenate utilization dramatically alters the susceptibility of yeast cells to ergosterol biosynthesis inhibitors. Genome-wide transcription and MS-based analyses revealed that this regulation is mediated by changes both in the expression of ergosterol biosynthesis genes and in the levels of sterol intermediates. Consistent with these findings, drug interaction experiments indicated that inhibition of pantothenic acid utilization synergizes with the activity of the ergosterol molecule-targeting antifungal amphotericin B and antagonizes that of the ergosterol pathway-targeting antifungal drug terbinafine. Our finding that CoA metabolism controls ergosterol biosynthesis and susceptibility to antifungals could set the stage for the development of new strategies to manage fungal infections and to modulate the potency of current drugs against drug-sensitive and -resistant fungal pathogens.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.

Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

The coenzyme A biosynthetic pathway: A new tool for prodrug bioactivation.

Prodrugs account for more than 5% of pharmaceuticals approved worldwide. Over the past decades several prodrug design strategies have been firmly established; however, only a few functional groups remain amenable to this approach. The aim of this overview is to highlight the use of coenzyme A (CoA) biosynthetic enzymes as a recently explored bioactivation scheme and provide information about its scope of utility. This emerging tool is likely to have a strong impact on future medicinal and biological studies as it offers promiscuity, orthogonal selectivity, and the capability of assembling exceptionally large molecules.

Neurodegeneration with Brain Iron Accumulation: Genetic Diversity and Pathophysiological Mechanisms.

Neurodegeneration with brain iron accumulation (NBIA) comprises a heterogeneous group of progressive disorders with the common feature of excessive iron deposition in the brain. Over the last decade, advances in sequencing technologies have greatly facilitated rapid gene discovery, and several single-gene disorders are now included in this group. Identification of the genetic bases of the NBIA disorders has advanced our understanding of the disease processes caused by reduced coenzyme A synthesis, impaired lipid metabolism, mitochondrial dysfunction, and defective autophagy. The contribution of iron to disease pathophysiology remains uncertain, as does the identity of a putative final common pathway by which the iron accumulates. Ongoing elucidation of the pathogenesis of each NBIA disorder will have significant implications for the identification and design of novel therapies to treat patients with these disorders.

Extracellular 4'-phosphopantetheine is a source for intracellular coenzyme A synthesis.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Antiplasmodial dihetarylthioethers target the coenzyme A synthesis pathway in Plasmodium falciparum erythrocytic stages.

BACKGROUND: Malaria is a widespread infectious disease that threatens a large proportion of the population in tropical and subtropical areas. Given the emerging resistance against the current standard anti-malaria chemotherapeutics, the development of alternative drugs is urgently needed. New anti-malarials representing chemotypes unrelated to currently used drugs have an increased potential for displaying novel mechanisms of action and thus exhibit low risk of cross-resistance against established drugs. RESULTS: Phenotypic screening of a small library (32 kinase-inhibitor analogs) against Plasmodium falciparum NF54-luc asexual erythrocytic stage parasites identified a diarylthioether structurally unrelated to registered drugs. Hit expansion led to a series in which the most potent congener displayed nanomolar antiparasitic activity (IC50 = 39 nM, 3D7 strain). Structure-activity relationship analysis revealed a thieno[2,3-d]pyrimidine on one side of the thioether linkage as a prerequisite for antiplasmodial activity. Within the series, the oxazole derivative KuWei173 showed high potency (IC50 = 75 nM; 3D7 strain), good solubility in aqueous solvents (1.33 mM), and >100-fold selectivity toward human cell lines. Rescue experiments identified inhibition of the plasmodial coenzyme A synthesis as a possible mode of action for this compound class. CONCLUSIONS: The class of antiplasmodial bishetarylthioethers reported here has been shown to interfere with plasmodial coenzyme A synthesis, a mechanism of action not yet exploited for registered anti-malarial drugs. The oxazole congener KuWei173 displays double-digit nanomolar antiplasmodial activity, selectivity against human cell lines, high drug likeness, and thus represents a promising chemical starting point for further drug development.

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

UNLABELLED: 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. IMPORTANCE: 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.

Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.

The STM4195 gene product (PanS) transports coenzyme A precursors in Salmonella enterica.

UNLABELLED: Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of de novo pantothenate biosynthesis. In Salmonella enterica, precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the STM4195 gene product of Salmonella enterica serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC (dfp). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The STM4195 gene is renamed panS to reflect participation in pantothenate salvage that was uncovered herein. IMPORTANCE: This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in S. enterica that was distinct from the annotated role and led to its designation as PanS (pantothenate salvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this compound is present in the environment and may serve a role in CoA synthesis for some organisms. As such, this work raises important question about CoA salvage that can be pursued with future studies in bacteria and other organisms.

Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.

Coenzyme A (CoA) is a ubiquitous cofactor involved in numerous essential biochemical transformations, and along with its thioesters is a key regulator of intermediary metabolism. Pantothenate (vitamin B5) phosphorylation by pantothenate kinase (PanK) is thought to control the rate of CoA production. Pantothenate kinase associated neurodegeneration is a hereditary disease that arises from mutations that inactivate the human PANK2 gene. Aryl phosphoramidate phosphopantothenate derivatives were prepared to test the feasibility of using phosphopantothenate replacement therapy to bypass the genetic deficiency in the Pank1(-/-) mouse model. The efficacies of candidate compounds were first compared by measuring the ability to increase CoA levels in Pank1(-/-) mouse embryo fibroblasts. Administration of selected candidate compounds to Pank1(-/-) mice corrected their deficiency in hepatic CoA. The PanK bypass was confirmed by the incorporation of intact phosphopantothenate into CoA using triple-isotopically labeled compound. These results provide strong support for PanK as a master regulator of intracellular CoA and illustrate the feasibility of employing PanK bypass therapy to restore CoA levels in genetically deficient mice.

Amino-4-imidazolecarboxamide ribotide directly inhibits coenzyme A biosynthesis in Salmonella enterica.

Aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a by-product of histidine biosynthesis. In bacteria, yeast, and humans, accumulation of AICAR has been shown to affect an array of cellular processes by both direct and indirect mechanisms. In purine biosynthesis, AICAR is the substrate of the bifunctional protein phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase (PurH, EC 2.1.2.3/3.5.4.10). Strains lacking PurH accumulate AICAR and have a defect in the synthesis of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine. The formation of HMP is also compromised in vivo when coenzyme A (CoA) levels are reduced. Our results show that the in vivo accumulation of AICAR decreased total CoA pools and, further, that AICAR inhibited the activity of pantoate ß-alanine ligase in vitro (PanC, EC 6.3.2.1). These results demonstrated a mechanism of AICAR action and provide new insights into the metabolic consequences of disrupting purine metabolism.

An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in ß-alanine and coenzyme A biosynthesis.

ß-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. ß-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 ß-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 ß-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 ß-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 ß-alanine biosynthesis in these organisms.