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Synthetic active site probes for PKS and NRPS biosynthetic enzymes

  • Author(s): Meier, Jordan Leslie
  • et al.
Abstract

Polyketides and nonribosomal peptides constitute two classes of natural products which are well known for their application as antibiotic, antiparasitic, antifungal, and anticancer agents. One of the most remarkable discoveries yielded by studies into the biosynthesis of these compounds has been the finding that these seemingly disparate chemical structures are produced by a similar biosynthetic logic, in which multimodular enzymes activate, condense, and tailor a series of monomer units to produce the final natural product. The finding that the sequence and identity of active sites occurring in polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymes dictates the structure and biological activity of the natural product has lead to studies towards reengineering these synthases for the production of novel small molecules, an approach known as combinatorial biosynthesis. However, the many difficulties encountered in such studies emphasize the need for a greater knowledge of these biosynthetic systems at the protein level before the full potential of such approaches can be achieved. The goal of the work in this thesis was to develop and apply chemical probes capable of facilitating structural and proteomic studies of PKS and NRPS enzymes, two aspects of these systems which have proven problematic to traditional methods of analysis. For this purpose, a series of fluorescence, affinity, and crosslinking probes were synthesized and evaluated for their ability to label enzyme activities commonly utilized by PKS and NRPS enzymes. These studies lead to the development of a chemoenzymatic crosslinking approach capable of directly reporting on the protein-protein interactions of the acyl carrier protein-dehydratase pair found in PKS systems, as well as methods for the specific labeling of PKS and NRPS carrier protein, ketosynthase, thioesterase, and dehydratase domains with fluorescence and affinity reagents in complex proteomic mixtures. Finally, these chemical proteomic probes were integrated with a high- content, mass spectrometry based protein identification platform for the global proteomic analysis of PKS and NRPS biosynthesis in the model bacterium Bacillus subtilis. Through their continued application such methods have the potential to provide unique insights into PKS and NRPS biosynthetic pathways, facilitating the discovery and production of new therapeutic agents.

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