RESUMEN
No longer in-F-able: fluorine building blocks can be used in polyketide biosynthesis. This represents a more flexible approach to organofluorines than the traditional use of fluorinated starter units in multistep organic syntheses, and will hopefully increase the number of compounds available for drug development.
Asunto(s)
Productos Biológicos/metabolismo , Flúor/química , Biocatálisis , Productos Biológicos/química , Coenzima A/metabolismo , Halogenación , Malonatos/química , Malonatos/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/química , Policétidos/metabolismoRESUMEN
Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzyme's substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.
Asunto(s)
Aciltransferasas/metabolismo , Policétidos/metabolismo , Aciltransferasas/genética , Antibacterianos/química , Antibacterianos/metabolismo , Eritromicina/análogos & derivados , Eritromicina/biosíntesis , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Sintasas Poliquetidas/química , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Enzyme-directed mutasynthesis is an emerging strategy for the targeted derivatization of natural products. Here, data on the synthesis of malonic acid derivatives for feeding studies in Saccharopolyspora erythraea , the mutagenesis of DEBS and bioanalytical data on the experimental investigation of studies on the biosynthetic pathway towards erythromycin are presented.
RESUMEN
T7 RNA polymerase is an important biocatalyst that is used in diverse biotechnological applications such as in vitro transcription or protein expression. The enzyme displays high substrate specificity which is payed by significant limitations regarding incorporation of synthetic nucleotide analogs. Of specific interest is enzymatic synthesis of 2'-O-methyl-modified RNA as these nucleic acids exhibit improved biochemical and pharmacokinetic properties that make them attractive for diagnostic and therapeutic purposes. We report here on the development of an activity-based selection/screening approach for assessing polymerase activities in the presence of 2'-O-methyl-modified nucleotides, and on the identification of one variant T7 RNA polymerase which is capable of synthesizing all-2'-O-methyl RNA.