dephosphocoenzyme-a and 4--phosphopantetheine

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.

Topics: Brain; Cloning, Molecular; Coenzyme A; Escherichia coli; Exome; Female; Fibroblasts; Gene Expression Regulation; Humans; Iron; Male; Mitochondria; Mutation, Missense; Nerve Degeneration; Pantetheine; Pedigree; Phosphorylation; Saccharomyces cerevisiae; Transferases

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine (PhP) to form dephosphocoenzyme A. This reaction sits at the branch point between the de novo pathway and the salvage pathway, and has been shown to be a rate-limiting step in the biosynthesis of CoA. Importantly, bacterial and mammalian PPATs share little sequence homology, making the enzyme a potential target for antibiotic development. A series of steady-state kinetic, product inhibition, and direct binding studies with Mycobacterium tuberculosis PPAT (MtPPAT) was conducted and suggests that the enzyme utilizes a nonrapid-equilibrium random bi-bi mechanism. The kinetic response of MtPPAT to the binding of ATP was observed to be sigmoidal under fixed PhP concentrations, but substrate inhibition was observed at high PhP concentrations under subsaturating ATP concentrations, suggesting a preferred pathway to ternary complex formation. Negative cooperativity in the kinetic response of MtPPAT to PhP binding was observed under certain conditions and confirmed thermodynamically by isothermal titration calorimetry, suggesting the formation of an asymmetric quaternary structure during sequential ligation of substrates. Asymmetry in binding was also observed in isothermal titration calorimetry experiments with dephosphocoenzyme A and CoA. X-ray structures of MtPPAT in complex with PhP and the nonhydrolyzable ATP analogue adenosine-5'-[(α,β)-methyleno]triphosphate were solved to 1.57 Å and 2.68 Å, respectively. These crystal structures reveal small conformational changes in enzyme structure upon ligand binding, which may play a role in the nonrapid-equilibrium mechanism. We suggest that the proposed kinetic mechanism and asymmetric character in MtPPAT ligand binding may provide a means of reaction and pathway regulation in addition to that of the previously determined CoA feedback.

Topics: Adenosine Triphosphate; Calorimetry; Coenzyme A; Crystallography, X-Ray; Feedback, Physiological; Kinetics; Models, Biological; Models, Molecular; Mycobacterium tuberculosis; Nucleotidyltransferases; Pantetheine; Protein Conformation; Protein Structure, Quaternary; Recombinant Proteins; Thermodynamics

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. This study examines the role of binding interactions in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-CoA complex. These groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interaction. The changes in the Gibbs free energy of the enzyme-substrate complex (DeltaDeltaG(ES)) and enzyme-transition state complex (DeltaDeltaG) brought about by the modification were measured. Cases where DeltaDeltaG exceeds DeltaDeltaG(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis, we show that the H-bond interactions between the Gly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducing the energy barrier to catalysis by an additional 3.3 kcal/mol. Together, these binding interactions contribute approximately 10(6) to the k(cat)/K(m).

Topics: Acyl Coenzyme A; Binding Sites; Binding, Competitive; Catalysis; Coenzyme A; Enzyme Stability; Hydrolases; Kinetics; Ligands; Mutagenesis, Site-Directed; Pantetheine; Substrate Specificity; Thermodynamics

Weanling rats were fed a pantothenic acid (PA)-free diet for 11 days. Although the animals did not show symptoms of vitamin deficiency, the concentrations of total and free CoA (analyzed with 2-oxoglutarate dehydrogenase), the levels of CoA, dephospho-CoA and 4'-phosphopantetheine (assayed together in the N-acetylation reaction) were decreased. As PA deficiency developed (by days 33-44 of the experiment), the reduction of the content of these metabolites and short-chain acyl-CoA became more pronounced. The level of long-chain acyl-CoA, the ratios of free CoA/total CoA and long-chain acyl-CoA/total CoA remained unchanged. The coenzyme biosynthetic precursors demonstrated the most marked response to the severity of PA deficiency. The relative stability of the hepatocyte CoA pool is interpreted in terms of the cytosol ability to deposit the vitamin in the form of pantothenate-protein complexes.

Topics: Acyl Coenzyme A; Animals; Body Weight; Coenzyme A; Liver; Male; Pantetheine; Pantothenic Acid; Proteins; Rats

The transport of pantothenate by the rat small intestine occurs by simple diffusion. There was no significant difference in the rate of pantothenate absorption in the upper, middle or lower sections of the intestine. Coenzyme A was hydrolyzed to pantetheine and pantothenate in the intestinal lumen via the following series of reactions: coenzyme A leads to phosphopantetheine leads to pantetheine leads to pantothenate. Intestinal tissue, which contains high levels of pantetheinase, quickly degrades pantetheine to pantothenate, which is then transported to the blood and thence to other tissues. Tissue distribution patterns of 14C 5 hours after intraluminal administration of 14C-labeled coenzyme A or [14C]pantothenate were similar; approximately 40% of the 14C was present in muscle and 10% in liver.

Topics: Absorption; Animals; Carbon Radioisotopes; Coenzyme A; Coenzymes; Hydrolysis; Intestine, Small; Male; Pantetheine; Pantothenic Acid; Rats; Rats, Inbred Strains