RESUMEN
The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.
Asunto(s)
Metiltransferasas/química , Metiltransferasas/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Secuencia de Aminoácidos , Antifúngicos/metabolismo , Bacillus subtilis/genética , Sitios de Unión , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , ARN Ribosómico 23S/biosíntesis , ARN Ribosómico 23S/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
A method was developed for determination of the rate of undesired point mutations upon cloning of synthetic DNA. The method relies on cloning of an oligonucleotide(s) into the E. coli alkaline phosphatase gene inactivated due to a small deletion within the active site. The oligonucleotide adds back the deleted sequence, but simultaneously introduces a missense mutation at a critical position. The activity of the enzyme is restored only if there is a predefined sequence change within the codon specifying an essential residue of the active site. The clones carrying the reactivated gene are detected by colony color screening on plates. The method is fast and simple, does not require specialized equipment nor enzymatic reactions, although a separate oligonucleotide needs to be provided for each sequence change to be evaluated. The procedure allows for the use of crude extracts of oligonucleotides and distinguishes between different types of sequence changes.