RESUMEN
Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC(50) in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Compuestos Bicíclicos con Puentes/química , Inhibidores de Proteasas/química , Amidohidrolasas/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Cristalografía por Rayos X , Pruebas de Sensibilidad Microbiana , Inhibidores de Proteasas/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimologíaRESUMEN
Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/química , Pseudomonas aeruginosa/enzimología , Staphylococcus aureus/enzimología , Streptococcus pneumoniae/enzimología , Thermotoga maritima/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/clasificación , Aminopeptidasas/genética , Sitios de Unión , Cristalografía por Rayos X , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genética , Electricidad Estática , Streptococcus pneumoniae/genética , Thermotoga maritima/genéticaRESUMEN
Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.