RESUMEN
IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7â Å resolution, respectively. The structure of the E.â coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.
Asunto(s)
Antibacterianos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Eritritol/análogos & derivados , Proteínas de Escherichia coli/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Oxidorreductasas/química , Fosfatos de Azúcar/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Eritritol/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismoRESUMEN
The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate-free LytB has a special [4Fe-4S](2+) cluster with a yet unsolved structure. This motivated us to use synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical-molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate-free LytB. The apical iron atom of the [4Fe-4S](2+) is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) and to the inhibitors (E)-4-amino-3-methylbut-2-en-1-yl diphosphate and (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Terpenos/metabolismo , Vías Biosintéticas , Cristalografía por Rayos X , Difosfatos/química , Difosfatos/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear BiomolecularRESUMEN
The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.
Asunto(s)
Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/enzimología , Proteínas Bacterianas/química , Eritritol/análogos & derivados , Hemiterpenos/química , Complejos Multienzimáticos/metabolismo , Compuestos Organofosforados/química , Compuestos Organofosforados/síntesis química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfatos de Azúcar/síntesis química , Agrobacterium tumefaciens/metabolismo , Catálisis , Pruebas de Enzimas , Eritritol/síntesis química , Eritritol/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Complejos Multienzimáticos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Especificidad por Sustrato , Fosfatos de Azúcar/químicaRESUMEN
The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Eritritol/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Fosfatos de Azúcar/farmacología , Terpenos/metabolismo , Biocatálisis , Vías Biosintéticas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Eritritol/análogos & derivados , Eritritol/química , Proteínas de Escherichia coli/aislamiento & purificación , Cinética , Modelos Moleculares , Estructura Molecular , Oxidorreductasas/aislamiento & purificación , Relación Estructura-Actividad , Fosfatos de Azúcar/síntesis química , Fosfatos de Azúcar/química , Terpenos/químicaRESUMEN
[reaction: see text] The concecutive Criegee rearrangement reactions were studied for tert-butyl trifluoroacetate, triarylcarbinols, and benzophenone ketales with trifluoroperacetic acid (TFPAA) in trifluoroacetic acid (TFA). The formation of methyl acetate and methyl trifluoroacetate indicates that the consecutive double-O-insertion process has taken place for tert-butyl trifluoroacetate. The intermediate dimethoxymethylcarbonium ion was detected below 5 degrees C. A consecutive triple-O-insertion process has been observed for triarylmethanols and benzophenone ketals. A new high yield method of corresponding diaryl carbonates synthesis was developed.