Bacterial type I polyketide synthases (PKSs) are mega-enzyme assembly lines responsible for generating the macrolactone core of a wide range of polyketide products that are biologically active natural compounds (e.g. antimicrobial, antifungal, antiviral, anticancer, and immunosuppressant compounds). Indicative of their significance, polyketide natural products currently form the basis for nearly one-third of pharmaceuticals. PKSs employ a modular multi-step mechanism to produce polyketides, and bioengineering these systems has immense potential for the creation of new chemotypes with invaluable applications in drug discovery. However, such efforts have met with limited success, reflecting our poor structural and mechanistic understanding of the modular process to generate polyketides. We recently employed cryo-electron microscopy (cryo-EM) to show the first subnanometer resolution structures of the full length PikAIII module from the pikromycin PKS biosynthetic pathway, a prototype for assembly-line PKS systems. The findings not only revealed an unexpected module architecture undergoing extensive structural rearrangements, but also showed that the type of substrate linked to a highly mobile acyl carrier protein (ACP) domain specifies its positioning in a way that facilitates assembly-line throughput. By employing recent breakthroughs in cryo-EM technologies, we now aim to obtain high resolution cryo-EM structures of PikAIII and of the terminal PikAIV module, in an effort to resolve several lingering question regarding functional interfaces and module dynamics. Our studies will include both natural and unnatural substrates, seeking to reveal the principles of substrate recognition and processing in these remarkable macromolecular factories. The findings from the proposed studies will for the basis for renewed bioengineering efforts towards the creation of PKSs that can efficiently produce novel compounds of high medicinal value.

Public Health Relevance

Type I modular polyketide synthases (PKSs) are enzyme assembly lines for the synthesis of chemically diverse polyketide natural products that form the basis for nearly one-third of pharmaceuticals (e.g. antimicrobial, antifungal, antiviral, anticance, and immunosuppressant compounds). We propose to apply cryo-electron microscopy (cryo-EM) analysis in order to obtain high-resolution structures of PikAIII and PikAIV, two model PKS modules, at different stages of their catalytic cycle and while processing natural and unnatural substrates. The elucidation of structural and mechanistic details of substrate recognition and processing by PKSs will be invaluable for the design of new PKS modules that create novel molecules with high medicinal value.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gerratana, Barbara
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Drake, Eric J; Miller, Bradley R; Shi, Ce et al. (2016) Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529:235-8
Smith, Janet L; Skiniotis, Georgios; Sherman, David H (2015) Architecture of the polyketide synthase module: surprises from electron cryo-microscopy. Curr Opin Struct Biol 31:9-19