RESUMEN
The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of ß-ketoacyl-ACP substrates to ß-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the Mycobacterium smegmatis genome uncovered several putative FabG-like proteins. Among them, we identified M. smegmatis MSMEG_6753 whose gene was found adjacent to MSMEG_6754, encoding a recently characterized enoyl-CoA dehydratase, and to MSMEG_6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a fabG thermosensitive Escherichia coli mutant. Furthermore, comparison of the apo and the NADP+-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A ΔMSMEG_6753 deletion mutant could be generated in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.
Asunto(s)
Acilcoenzima A/química , Oxidorreductasas de Alcohol/química , Proteínas Bacterianas/química , Mycobacterium smegmatis/química , Mycobacterium tuberculosis/química , NADP/química , Acilcoenzima A/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Enoil-CoA Hidratasa/química , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Prueba de Complementación Genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , NADP/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The pharmacologic effect of an antibiotic is directly related to its unbound concentration at the site of infection. Most commercial antibiotics have been selected in part for their low propensity to interact with serum proteins. These nonspecific interactions are classically evaluated by measuring the MIC in the presence of serum. As higher-throughput technologies tend to lose information, surface plasmon resonance (SPR) is emerging as an informative medium-throughput technology for hit validation. Here we show that SPR is a useful automatic tool for quantification of the interaction of model antibiotics with serum proteins and that it delivers precise real-time kinetic data on this critical parameter.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Sanguíneas/metabolismo , Resonancia por Plasmón de Superficie/métodos , Unión ProteicaRESUMEN
Ensuring the availability of new antibiotics to eradicate resistant pathogens is a critical issue, but very few new antibacterials have been recently commercialized. In an effort to rationalize their discovery process, the industry has utilized chemical library and high-throughput approaches already applied in other therapeutical areas to generate new antibiotics. This strategy has turned out to be poorly adapted to the reality of antibacterial discovery. Commercial chemical libraries contain molecules with specific molecular properties, and unfortunately systemic antibacterials are more hydrophilic and have more complex structures. These factors are critical, since hydrophobic antibiotics are generally inactive in the presence of serum. Here, we review how the skewed distribution of systemic antibiotics in chemical space influences the discovery process.