RESUMEN
Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation-PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Modelos Moleculares , Complejos Multienzimáticos/química , Péptido Sintasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Biocatálisis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Biología Computacional , Secuencia Conservada , Bases de Datos de Proteínas , Enterobactina/biosíntesis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lisina/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Prolina/química , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Programas InformáticosRESUMEN
A 7-year-old boy presented to the emergency department with fever, cough, congestion, abdominal pain, myalgias, and morbilliform rash. Several aspects of the patient's history, including recent travel, living on a farm, exposure to sick contacts, and new medications, resulted in a wide differential diagnosis. Initial laboratory testing revealed leukocytosis with neutrophilia and elevated atypical lymphocytes, but did not reveal any infectious causes of illness. He was discharged from the hospital, but then represented to the emergency department a day later with worsening rash, continued fever, abdominal pain, and poor intake. He was then admitted. A more comprehensive laboratory evaluation was initiated. During this hospital course, the patient's physical examination changed when he developed head and neck edema, and certain laboratory trends became clearer. With the assistance of several specialists, the team was able to reach a more definitive diagnosis and initiate treatment to appropriately manage his condition.