RESUMO
The concept of combinatorial biosynthesis promises access to compound libraries based on privileged natural scaffolds. Ever since the elucidation of the biosynthetic pathway towards the antibiotic erythromycin A in 1990, the predictable manipulation of type I polyketide synthase megaenzymes was investigated. However, this goal was rarely reached beyond simplified model systems. In this study, we identify the intermediates in the biosynthesis of the polyether monensin and numerous mutated variants using a targeted metabolomics approach. We investigate the biosynthetic flow of intermediates and use the experimental setup to reveal the presence of selectivity filters in polyketide synthases. These obstruct the processing of non-native intermediates in the enzymatic assembly line. Thereby we question the concept of a truly modular organization of polyketide synthases and highlight obstacles in substrate channeling along the cascade. In the search for the molecular origin of a selectivity filter, we investigate the role of different thioesterases in the monensin gene cluster and the connection between ketosynthase sequence motifs and incoming substrate structures. Furthermore, we demonstrate that the selectivity filters do not apply to new-to-nature side-chains in nascent polyketides, showing that the acceptance of these is not generally limited by downstream modules.
Assuntos
Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Engenharia de Proteínas , Policetídeos/química , Conformação ProteicaRESUMO
The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5mon ) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5mon , insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.
Assuntos
Produtos Biológicos/síntese química , Monensin/análogos & derivados , Monensin/síntese química , Policetídeo Sintases/química , Aciltransferases/química , Biologia Computacional , Escherichia coli/metabolismo , Fermentação , Malonatos/química , Modelos Moleculares , Monensin/farmacologia , Conformação Proteica , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Although protein kinase inhibitors present excellent pharmaceutical opportunities, lack of selectivity and associated therapeutic side effects are common. Bisubstrate-based inhibitors targeting both the high-selectivity peptide substrate binding groove and the high-affinity ATP pocket address this. However, they are typically large and polar, hampering cellular uptake. This paper describes a modular development approach for bisubstrate-based kinase inhibitors furnished with cell-penetrating moieties and demonstrates their cellular uptake and intracellular activity against protein kinase C (PKC). This enzyme family is a longstanding pharmaceutical target involved in cancer, immunological disorders, and neurodegenerative diseases. However, selectivity is particularly difficult to achieve because of homology among family members and with several related kinases, making PKC an excellent proving ground for bisubstrate-based inhibitors. Besides the pharmacological potential of the novel cell-penetrating constructs, the modular strategy described here may be used for discovering selective, cell-penetrating kinase inhibitors against any kinase and may increase adoption and therapeutic application of this promising inhibitor class.