RESUMEN
A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Antimaláricos/farmacología , Fosfomicina/análogos & derivados , Hemiterpenos , Malaria/tratamiento farmacológico , Complejos Multienzimáticos/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Pentosafosfatos/metabolismo , Plasmodium falciparum/efectos de los fármacos , Terpenos/farmacología , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Fosfomicina/farmacología , Genes Protozoarios , Malaria/parasitología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Ácido Mevalónico/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , Compuestos Organofosforados/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Eritritol/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ácido Mevalónico/metabolismo , Oxidorreductasas , Fosfatos de Poliisoprenilo/biosíntesis , Fosfatos de Azúcar/metabolismo , Animales , Proteínas Bacterianas/genética , Eritritol/análogos & derivados , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Genes Bacterianos/genética , Genes Esenciales/genética , Prueba de Complementación Genética , Humanos , Fosfatos de Poliisoprenilo/metabolismo , Homología de SecuenciaRESUMEN
Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli. The purified P. aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method. A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P. aeruginosa DXP synthase. Fluoropyruvate is described as a first inhibitor of DXP synthase.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fosfomicina/análogos & derivados , Complejos Multienzimáticos/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Piruvatos/farmacología , Transferasas/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/genética , Fosfomicina/farmacología , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pseudomonas aeruginosa/genética , Espectrofotometría/métodos , Transferasas/genética , Transferasas/metabolismoRESUMEN
Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.
Asunto(s)
Virus de la Diarrea Viral Bovina/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Ribosomas/metabolismo , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Plásmidos , Proteína de Unión al Tracto de Polipirimidina , Conejos , Espectrofotometría UltravioletaRESUMEN
The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.
Asunto(s)
Antimaláricos/uso terapéutico , Fosfomicina/uso terapéutico , Malaria/tratamiento farmacológico , Profármacos/uso terapéutico , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Animales , Antimaláricos/química , Modelos Animales de Enfermedad , Fosfomicina/análogos & derivados , Fosfomicina/química , Ratones , Complejos Multienzimáticos/antagonistas & inhibidores , Organofosfonatos/química , Oxidorreductasas/antagonistas & inhibidores , Plasmodium/efectos de los fármacos , Profármacos/síntesis química , Profármacos/química , Profármacos/metabolismo , Resultado del TratamientoRESUMEN
In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.