No abstract

Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli ,B-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular f.-alanine-derived metabolites from cells grown on increasing 8l-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to ,B-alanine input, whereas the CoA content increased as a function of external ,B-alanine concentration, reaching a maximum at 8 ,M ,B-alanine. Further increase in the fl-alanine concentration led

Pantothenate kinase (PANK) is a metabolic enzyme that regulates cellular coenzyme A (CoA) levels. There are three human PANK genes, and inactivating mutations in PANK2 lead to pantothenate kinase associated neurodegeneration (PKAN). Here we performed a library screen followed by chemical optimization to produce PZ-2891, an allosteric PANK activator that crosses the blood brain barrier. PZ-2891 occupies the pantothenate pocket and engages the dimer interface to form a PANK•ATP•Mg2+•PZ-2891 complex. The binding of PZ-2891 to one protomer locks the opposite protomer in a catalytically active conformation that is refractory to acetyl-CoA inhibition. Oral administration of PZ-2891 increases CoA levels in mouse liver and brain. A knockout mouse model of brain CoA deficiency exhibited weight loss, severe locomotor impairment and early death. Knockout mice on PZ-2891 therapy gain weight, and have improved locomotor activity and life span establishing pantazines as novel therapeutics for the treatment of PKAN.

Escherichia coli 0-alanine auxotrophs (panD2) were used to manipulate the specific cellular content of coenzyme A (CoA) and assess the associated physiological effects. Growth-limiting concentrations of CoA resulted in an increase in phospholipid/protein ratio in reUA mutants, but not in their rel+ counterparts4 indicating that protein biosynthesis was more severely affected by CoA deprivation than phospholipid biosynthesis. Acetyl-CoA was the dominant component (79.8%) of the CoA pool in cells exponentially growing in glucose-minimal medium, with significant concentrations of CoA (13.8%) and succinyl-CoA (5.9%) also detected. Malonyl-CoA was a minor species (0.5%), and the mixed digulfide of CoA and glutathione was not present. Acetyl-CoA was also the major constituent in cells depleted of CoA. On the other hand, succinyl-CoA was absent, suggesting that the protein synthesis defect may be due to the inability to generate sufficient quantities of precursors via the tricarboxylic acid cycle to support amino acid biosynthesis. PIroduction of acyl carrier protein was controlled in part by the availability of CoA, and the lower concentration of acyl carrier protein in CoA-depleted cells was associated with a concomitant decrease in the saturated/unsaturated fatty acid ratio.Coenzyme A (CoA) and acyl carrier protein (ACP) are the major cellular constituents in Escherichia coli that are biosynthesized from the vitatnii pantothenic acid (1,22). Both compounds function as coenzymes in the metabolism of acyl moieties which are bound as thioesters to the terminal sulfhydryl of the 4'-phosphopantetheine portion of these molecules. These two cofactors are interrelated, in that CoA serves as the donor of 4'-phosphopantetheine to apo-ACP (8), and the prosthetic group of ACP turns over (13,21), thus releasing 4'-phosphopantetheine that is either reused for CoA biosynthesis (14, 21) or irreversibly excreted from the cell (14). Several investigators have observed that the intracellular concentration of CoA can be manipulated by varying the amount of pantothenate (1, 11) or 3-alanine (11,12,14,21) Showe and DeMoss (24). Cells were washed once with 50 mM Tris hydrochloride, pH 7.5, and 1 mM MgCl2 and then resuspended in 0.4 ml of the same buffer containing 100 ,ug of lysozyme per ml and 4 ,ug DNase I per ml. The suspension was freeze-thawed three times with a dry ice and ethanol bath and a 37°C water bath, followed by sonication in a cuphorn attached to a Heat Systems sonicator for three 10-s intervals at an output setting of 10. The resulting sample was then centrifuged for 15 min in a Beckman Microfuge-12 (Beckman Instruments, Inc., Fullerton, Calif.). The pellet contained less than 2% of the ACP plus CoA in the total extract. The amount of CoA and ACP,in the P-alanine-labeled cell extracts was determined by thin-layer chromatography (11).Protein was determined by using the microbiuret method (20), with bovine serum albumin as the standard. Cell pellets were dissolved in 1 N NaOH at 60°C prior to protein measurements. Phosphol...

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl–coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl–coenzyme A synthetase and acyl–coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.