This study identifies that Coenzyme A covalently modifies specific cysteine residues on a wide range of proteins, a process termed protein CoAlation. In Staphylococcus aureus, mass spectrometry analysis revealed 356 CoAlated proteins. Specific targets identified include:
- Glyceraldehyde-3-phosphate dehydrogenase (SaGAPDH), modified at the catalytic Cys151 [1].
- Transcriptional regulators: SarR, CtsR, AgrA (CoAlated on Cys6 and Cys199), PerR (CoAlated on Cys142), and SarS [1].
- Antioxidant proteins: thioredoxin (Trx), alkyl hydroperoxide reductase C (AhpC), thiol peroxidase (Tpx, CoAlated on active site Cys60), malate: quinone oxidoreductases 1 and 2 (Mqo1/2), and others [1].
- Many ribosomal proteins, including L12, S12, L14, S18, L32, L33 (RpmG3), and L36 (RpmJ) [1].
- Numerous metabolic enzymes: succinate-CoA ligase, acyl-CoA ligase, HMG-CoA synthase, acetyl-CoA carboxylase, acyl-CoA dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase 2 (GapA2), pyruvate kinase (Pyk), ATP-dependent 6-phosphofructokinase (PfkA), acetate kinase (AckA), alcohol dehydrogenase (Adh), and others [1].
Coenzyme A itself is not a drug targeting specific receptors but rather an obligate cofactor for hundreds of enzymes in intermediary metabolism. Its primary molecular targets include acyl-CoA synthetases, carnitine O-acetyltransferase, citrate synthase, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), among many others. CoA also binds to the acyl-CoA-binding protein (ACBP), which serves as an intracellular carrier and pool former for acyl-CoA esters. In addition, CoA acts as the source of the 4′-phosphopantetheine prosthetic group for carrier proteins in fatty acid, polyketide, and nonribosomal peptide synthases.

- In vitro CoAlation of SaGAPDH: Recombinant His-tagged SaGAPDH was incubated with 2 mM CoA disulfide (CoASSCoA). This treatment resulted in a 90% inhibition of its enzymatic activity. The inhibitory effect was completely reversed by the addition of 10 mM dithiothreitol (DTT) [1].
- Protection against overoxidation: In vitro CoAlation of SaGAPDH before exposure to 10 mM hydrogen peroxide (H₂O₂) resulted in nearly 100% recovery of its enzymatic activity upon subsequent treatment with 10 mM DTT. This indicates that CoAlation protects the catalytic Cys151 from irreversible overoxidation [1].
- Inhibition by H₂O₂: Exposure of recombinant SaGAPDH to 10 µM H₂O₂ resulted in an approximate 50% decrease in its catalytic activity. A concentration of 1 mM H₂O₂ caused 95% inhibition, while 10 mM H₂O₂ completely blocked its activity. The inactivation by 10 mM H₂O₂ was only partially reversible, with only 40% of activity recovered by 10 mM DTT [1].

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In vitro studies demonstrate that CoA is an essential cofactor for numerous enzymatic reactions, including the formation of acetyl-CoA from pyruvate, fatty acid oxidation, and the citric acid cycle. In cell culture models, extracellular CoA does not elevate intracellular CoA levels, as it is quantitatively degraded to pantothenate in the presence of cells. For example, when CoA (10 µM) was incubated with C3A cells for 20 hours, it was completely converted to pantothenate, indicating that cells do not directly take up exogenous CoA but rather utilize its degradation products for de novo synthesis. CoA has also been shown to modulate AMPK activity, with palmitoyl-CoA activating AMPK in an in vitro kinase assay.

- Induction in bacteria: Protein CoAlation occurs at a low basal level in Gram-negative (E. coli) and Gram-positive (B. megaterium, S. aureus) bacteria under normal growth conditions. It is strongly induced in response to treatment with oxidizing agents, including 10-100 mM hydrogen peroxide (H₂O₂), 2 mM diamide, and 100-150 µM sodium hypochlorite (NaOCl) for 30 minutes [1].
- Induction by metabolic stress: Glucose starvation (transferring bacteria to medium lacking glucose for 60-120 minutes) strongly induces protein CoAlation in E. coli, B. megaterium, and S. aureus. This CoAlation is reversed by the re-addition of 20 mM glucose for 30 minutes [1].
- Reversibility: Diamide-induced protein CoAlation in E. coli, B. megaterium, and S. aureus is a reversible post-translational modification [1].
- In vivo CoAlation of SaGAPDH: In E. coli overexpressing His-SaGAPDH, treatment with 2 mM diamide induced strong CoAlation of the protein. The strongest CoAlation was observed in response to 100 µM NaOCl, while a weak signal was detected in cells treated with 10 mM H₂O₂ [1].

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In vivo, CoA plays a critical role in brain energy metabolism, fatty acid oxidation, and neurotransmitter synthesis. In a mouse model of pantothenate kinase-associated neurodegeneration (PKAN), brain CoA deficiency is associated with movement dysfunction, and restoration of brain CoA levels via treatment with the pantothenate kinase activator BBP-671 improves locomotion and survival. The Pank1fl/fl,Pank2fl/fl SynCre+ neuronal knockout mouse exhibits durable brain CoA deficiency and clear movement dysfunction, demonstrating the essential role of CoA in neuronal function. In Yucatan microswine, topical application of 4-hydroxyretinoic acid—a metabolite dependent on CoA-mediated acyl transfer—induces epidermal hyperplasia.

- GAPDH Enzymatic Assay: Recombinant SaGAPDH activity was determined by measuring the absorbance change at 340 nm and 25°C resulting from the production of NADH. The reaction was carried out in a 150 µl assay mixture containing 20 mM Tris-HCl (pH 8.7), 0.36 µM SaGAPDH, 1.25 mM NAD⁺, 1.25 mM EDTA, and 15 mM sodium arsenate. The reaction was started by the addition of 0.25 mM glyceraldehyde 3-phosphate. Initial reaction rates were calculated by determining the slope in the linear part of the curve during the first 80 seconds of the reaction. For inactivation experiments, SaGAPDH was preincubated with 1 µM, 10 µM, 100 µM, or 10 mM H₂O₂ for 10 minutes or with 10 mM CoASSCoA for 30 minutes before measuring remaining activity [1].
- Nudix 7 Hydrolase Assay: Recombinant His-Nudix 7 hydrolase (1.7 µg) was incubated in a total volume of 100 µl containing 50 mM (NH₄)HCO₃ and 0.2 mM CoASSG (CoA-glutathione mixed disulfide) at 37°C for 20 minutes with or without 5 mM MgCl₂. Reaction products and substrates were analyzed by HPLC, with elution monitored at 205 nm [1].

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A typical non-cellular assay for CoA involves measuring its concentration or enzymatic activity using biochemical kits. For example, the Coenzyme A Assay Kit (ab102504) utilizes a multi-step enzymatic reaction where free CoA is specifically converted to generate products that react with an OxiRed probe, producing color (λ = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The assay has a detection range of 2.5–250 µM and sensitivity >2.5 µM. Alternatively, CoA and its thioester derivatives can be quantified using LC-MS/MS with stable isotope-labeled internal standards generated via SILEC (Stable Isotope Labeling by Essential Nutrients in Cell Culture). For enzyme activity assays, CoA is typically incubated with the target enzyme, appropriate substrates (e.g., acetyl-CoA for acetyltransferase), and buffer conditions optimized for the specific reaction.

- Bacterial culture and treatment for protein CoAlation analysis: Bacterial species (E. coli, B. megaterium, S. aureus) were grown overnight in rich medium (Luria Bertani for E. coli and S. aureus; Nutrient Broth 3 for B. megaterium). Overnight cultures were diluted 1:100 in the same media and incubated until an optical density of 0.7 at 600 nm (OD₆₀₀). Cells were then treated with or without oxidizing agents for 30 minutes at 37°C: hydrogen peroxide (10 and 100 mM), diamide (2 mM), and NaOCl (150 µM). To induce metabolic stress, bacterial cultures at OD₆₀₀ of 0.7 were harvested by centrifugation and resuspended in M9 minimal medium supplemented with or without glucose as a source of carbohydrate [1].
- Cell lysis and protein extraction: For E. coli and B. megaterium, the pellet was resuspended in buffer containing 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 100 mM NEM (to prevent in vitro modification), and protease inhibitors. SDS was added to a final concentration of 1%, and the homogenate was sonicated before centrifugation at 21,000g for 10 minutes at room temperature. For S. aureus, the pellet was resuspended in a similar buffer, and lysostaphin (22 U/ml) was added to solubilize cell wall proteins, followed by incubation at 37°C for 30 minutes before SDS addition and sonication [1].

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In vitro cellular assays for CoA typically involve culturing cells in medium containing dialyzed or charcoal-stripped fetal bovine serum to eliminate unlabeled pantothenate contamination. To generate isotopically labeled CoA standards, cells (e.g., Hepa 1c1c7 mouse hepatoma cells or Drosophila S2 cells) are passaged 3–5 times in medium supplemented with [13C315N]-pantothenate (1 mg/L) instead of unlabeled pantothenate. After 3 passages, approximately 98–99% of cellular CoA species become labeled. For CoA quantification, cells are lysed, and the lysate is analyzed using LC-neutral loss scan of m/z = 507, which detects the characteristic fragmentation pattern of CoA derivatives. Cellular CoA levels can be modulated by adding specific fatty acids (e.g., propionate to generate propionyl-CoA) or by subjecting cells to various metabolic perturbations.

In vivo animal studies for CoA often utilize the Pank1fl/fl,Pank2fl/fl SynCre+ mouse model of pantothenate kinase-associated neurodegeneration (PKAN), which exhibits brain CoA deficiency and movement dysfunction. Animals are typically dosed daily via oral gavage with test compounds (e.g., the pantothenate kinase activator BBP-671) at doses ranging from 0.1 to 30 mg/kg for 7 days to assess pharmacokinetic and pharmacodynamic properties. Tissue samples (plasma, liver, brain, and cerebrospinal fluid) are collected at specified time points (e.g., 4 hours after the last dose) and analyzed for total CoA concentration using LC-MS/MS. Endpoints include measurement of CoA levels in blood, liver, and brain, as well as assessment of motor function (locomotion) and body weight changes.

Coenzyme A itself is not administered as a therapeutic agent due to its poor oral bioavailability and rapid degradation. Exogenous CoA is quantitatively degraded to pantothenate in the presence of cells, as demonstrated in C3A cell culture studies where CoA (10 µM) incubated with cells for 20 hours was completely converted to pantothenate. In the body, CoA is synthesized endogenously from pantothenate (vitamin B5) through a five-step enzymatic pathway: pantothenate → 4′-phosphopantothenate → 4′-phosphopantetheine → dephospho-CoA → CoA. The intracellular concentration of CoA is tightly regulated via feedback inhibition of pantothenate kinase by acyl-CoAs. Tissue CoA levels vary significantly; for example, E. coli growing on glucose has a CoA pool of approximately 400 µM, whereas bacteria growing on amino acids have about 100 µM.

According to safety data sheets, Coenzyme A is classified as causing skin irritation (Category 2), serious eye irritation (Category 2), and specific target organ toxicity – single exposure (Category 3), with potential to cause respiratory irritation. Precautionary measures include wearing protective gloves, eye protection, and working in a well-ventilated area. In animal studies, CoA itself has not been reported to cause significant systemic toxicity, but abnormalities in CoA biosynthesis are associated with human diseases. For instance, mutations in the PANK2 gene, which encodes pantothenate kinase 2, lead to pantothenate kinase-associated neurodegeneration (PKAN), a rare autosomal recessive disorder characterized by dystonia, parkinsonism, and premature death. While CoA is generally recognized as safe at physiological levels, high doses or prolonged exposure may cause local irritation at the site of contact.

[1]. Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells. Biochem J. 2018 Jun 6;475(11):1909-1937.
[2]. https://pubchem.ncbi.nlm.nih.gov/compound/87642

- Background on CoA thiols: CoA has been found to form CoA disulfides (CoASSCoA) and mixed disulfides with other low-molecular-weight thiols (e.g., CoA-cysteine and CoA-glutathione, CoASSG). The CoASSG heterodimer has been isolated from bacteria, yeast, human myocardial tissue, and parathyroid glands. Potent vasoconstrictive and proliferative effects of CoASSG were observed in cultured vascular smooth muscle cells, and it was also shown to inhibit the activity of bacterial RNA polymerase [1].
- Mechanism of protein CoAlation: When a cysteine thiol group is oxidized by ROS to an unstable sulfenic acid intermediate, it can react with nearby thiols, leading to mixed disulfides with LMW thiols like CoA. Formed disulfides are reversible regulatory events and function to protect unstable sulfenic acids against overoxidation to sulfinic and sulfonic acids [1].
- Role in oxidative stress: The study concludes that protein CoAlation is a widespread redox-regulated post-translational modification in bacteria, with a potential to protect critical reactive cysteines (e.g., in SaGAPDH) against irreversible overoxidation under oxidative stress conditions like exposure to H₂O₂ [1].
- MD simulation model: Molecular dynamics simulations proposed a "double anchor model" for GAPDH CoAlation. In the absence of NAD⁺, the ADP moiety of CoA occupies the vacant nucleotide-binding pocket in the oxidized form of GAPDH, positioning the CoA thiol group in the flexible pantetheine tail in close vicinity for covalent disulfide bond formation with catalytic Cys151 [1].

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Coenzyme A is a thiol composed of a pantothenic acid unit linked to a 3',5'-adenosine diphosphate unit via a phosphate anhydride bond and containing an aminoethanethiol unit. It is a metabolite in both E. coli and mice, and is also a coenzyme. Functionally, it is associated with ADP. It is the conjugate acid of coenzyme A(4-). Coenzyme A (CoA, CoASH, or HSCoA) is a coenzyme known for its role in fatty acid synthesis and oxidation, as well as in the oxidation of pyruvate in the citric acid cycle. All genomes sequenced to date encode enzymes that use coenzyme A as a substrate, and approximately 4% of cellular enzymes use coenzyme A or its thioester forms as substrates. It is used as an adjunct in acne treatment. Coenzyme A is present in or produced by E. coli (K12 strain, MG1655 strain). Coenzyme A has also been reported in fruit flies, humans, and other organisms with relevant data. Coenzyme A is a coenzyme containing pantothenic acid, adenosine 3-phosphate, adenosine 5-pyrophosphate, and cysteine; it participates in acyl transfer, especially in transacetylation reactions. See also: ... (See more...)

C21H36N7O16P3S

767.53

767.115

C, 32.86; H, 4.73; N, 12.77; O, 33.35; P, 12.11; S, 4.18

85-61-0

Coenzyme A trilithium;18439-24-2;Coenzyme A sodium;55672-92-9

87642

White to light yellow solid powder

2.0±0.1 g/cm3

146 - 147

-5ºC

1.737

-4.02

10

21

18

48

1270

5

CC(COP(O)(OP(O)(OC[C@](O1)([H])[C@](OP(O)(O)=O)([H])[C@](O)([H])[C@]1([H])N2C=NC(C2=NC=N3)=C3N)=O)=O)(C(O)([H])/C(O)=N/CC/C(O)=N/CCS)C

RGJOEKWQDUBAIZ-IBOSZNHHSA-N

InChI=1S/C21H36N7O16P3S/c1-21(2,16(31)19(32)24-4-3-12(29)23-5-6-48)8-41-47(38,39)44-46(36,37)40-7-11-15(43-45(33,34)35)14(30)20(42-11)28-10-27-13-17(22)25-9-26-18(13)28/h9-11,14-16,20,30-31,48H,3-8H2,1-2H3,(H,23,29)(H,24,32)(H,36,37)(H,38,39)(H2,22,25,26)(H2,33,34,35)/t11-,14-,15-,16+,20-/m1/s1

[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(3R)-3-hydroxy-2,2-dimethyl-4-oxo-4-[[3-oxo-3-(2-sulfanylethylamino)propyl]amino]butyl] hydrogen phosphate

coenzyme A; Coenzyme A (free acid); CoA; CoASH; CoA-SH; Zeel;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~125 mg/mL (~162.86 mM)
DMSO : ~100 mg/mL (~130.29 mM)

Solubility in Formulation 1: ≥ 1.63 mg/mL (2.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.63 mg/mL (2.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.63 mg/mL (2.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (32.57 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)