RESUMEN
Coenzyme A is an essential metabolite known for its central role in over one hundred cellular metabolic reactions. In cells, Coenzyme A is synthesized de novo in five enzymatic steps with vitamin B5 as the starting metabolite, phosphorylated by pantothenate kinase. Mutations in the pantothenate kinase 2 gene cause a severe form of neurodegeneration for which no treatment is available. One therapeutic strategy is to generate Coenzyme A precursors downstream of the defective step in the pathway. Here we describe the synthesis, characteristics and in vivo rescue potential of the acetyl-Coenzyme A precursor S-acetyl-4'-phosphopantetheine as a possible treatment for neurodegeneration associated with pantothenate kinase deficiency.
Asunto(s)
Trastornos Heredodegenerativos del Sistema Nervioso/tratamiento farmacológico , Panteteína/análogos & derivados , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Suero/química , Animales , Línea Celular , Modelos Animales de Enfermedad , Drosophila , Humanos , Ratones , Panteteína/administración & dosificación , Panteteína/síntesis química , Panteteína/aislamiento & purificación , Panteteína/farmacocinética , Resultado del TratamientoRESUMEN
Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-ß-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-ß-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-ß-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.
Asunto(s)
Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Biomimética , Mutagénesis Sitio-Dirigida , Panteteína/aislamiento & purificación , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Policétidos/química , Conformación ProteicaRESUMEN
FR901537 is a new aromatase inhibitor produced by a bacterium Bacillus sp. No. 3072. Structural studies of FR901537 suggested that it was a novel naphthol derivative having pantetheine in its structure. FR901537 showed a potent inhibitory activity against aromatase from human placenta or rat ovary, but did not inhibit the activity of 11 beta-hydroxylase from bovine adrenal cortex. Lineweaver-Burk plot analysis revealed that FR901537 is a competitive inhibitor.
Asunto(s)
Inhibidores de la Aromatasa , Naftoles/aislamiento & purificación , Naftoles/farmacología , Panteteína/análogos & derivados , Animales , Aromatasa/metabolismo , Bacillus , Bovinos , Femenino , Fermentación , Humanos , Ratones , Naftoles/química , Ovario/efectos de los fármacos , Ovario/enzimología , Panteteína/química , Panteteína/aislamiento & purificación , Panteteína/farmacología , Placenta/efectos de los fármacos , Placenta/enzimología , Ratas , Ratas WistarRESUMEN
Cyclosporin synthetase was isolated from a cyclosporin non-producing mutant of Beauveria nivea, strain YP 582. The enzyme has a molecular mass in the range of active cyclosporin synthetase and also contains 4'-phosphopantetheine as a prosthetic group. It is able to activate all constituent amino acids of cyclosporin A as thioesters and to carry out specific N-methylation reactions. Overall synthesis of the undecapeptide cyclosporin A in the presence of all necessary substrates was not observed, but the formation of the diketopiperazine cyclo-(D-alanyl-N-methyl-leucyl). This diketopiperazine represents a partial sequence of the cyclosporin molecule. It could be detected in the mycelium of the non-producing strain, whereas mycelium of the producing strain 7939/45 did not contain this compound. The results suggest that the inability of this mutant to produce cyclosporin A is caused by a mutation of the polypeptide chain of cyclosporin synthetase.