Polyketide natural products are the basis for a substantial number of pharmaceuticals, including some with newly discovered indications for inflammatory diseases. Development of polyketide lead compounds is challenging due to their chemical complexity and the inability to obtain most of them from natural sources. Thus the biosynthetic pathways are attractive targets for expanding chemical space through chemo-enzymatic synthesis and for engineering pathways to new function. The type I polyketide synthases (PKS) are assembly- line megasynthases where several distinct modules sequentially extend and modify a polyketide intermediate. This project examines fundamental aspects of PKS module function and of PKS enzyme domains that catalyze unusual reactions.
In Aim 1, a robust assay system using authentic, late-stage polyketide intermediates as substrates will be employed to determine the selectivity of PKS catalytic domains for their natural acyl carrier protein (ACP) partners and the limits of enzyme tolerance for foreign ACPs. Details of ACP-enzyme interaction will be visualized in crystal structures of crosslinked ACP-enzyme complexes and will aid future efforts to design of a broadly tolerated generic ACP.
In Aim 2, dehydratase domains will be investigated to determine the structural basis for two unusual activities: a rare dual dehydratase-isomerase activity and the unprecedented dehydration of ?-hydroxyacyl substrates.
In Aim 3, the structural basis for C-methyltransferase activity by domains embedded in PKS modules will be investigated and the substrate range will be explored. Macrolide antibiotics are among the most interesting polyketides, and a final aim will address the determinants of macrocycle formation by the thioesterases (TE) that offload polyketide products. Using four macrocycle- forming TEs of known structure and a panel of natural and non-natural substrates, Aim 4 will test the hypothesis that the active site of each TE is adapted to bind its natural substrate in conformations that are most conducive to cyclization and not hydrolysis to a linear product. Details of trapped acyl-enzyme intermediates will be visualized in crystal structures. These fundamental studies will have direct relevance to efforts to tap the amazing diversity of polyketide natural products by exploiting the biosynthetic pathways in development of new compounds. The enzymes that catalyze reactions of dehydration, isomerization, methyl transfer and macrocycle formation can be deployed as tools to expand the chemical potential of polyketide bio- activity.

Public Health Relevance

Microbes make a number of fascinating chemical compounds. The polyketide natural products are among the most interesting because they are the basis of many drugs. The polyketide biosynthetic machinery is a potential target for engineering to develop new drugs or improve existing ones. These studies will discover fundamental details about how complex biosynthetic machines function and will aid drug discovery efforts.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Sechi, Salvatore
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Skiba, Meredith A; Bivins, Marissa M; Schultz, John R et al. (2018) Structural Basis of Polyketide Synthase O-Methylation. ACS Chem Biol :
Skiba, Meredith A; Maloney, Finn P; Dan, Qingyun et al. (2018) PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production. Methods Enzymol 604:45-88
Tripathi, Ashootosh; Park, Sung Ryeol; Sikkema, Andrew P et al. (2018) A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit-Activating Enzyme CahJ. Chembiochem 19:1595-1600
Skiba, Meredith A; Sikkema, Andrew P; Moss, Nathan A et al. (2018) Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module. ACS Chem Biol 13:1640-1650
Dodge, Greg J; Maloney, Finn P; Smith, Janet L (2018) Protein-protein interactions in ""cis-AT"" polyketide synthases. Nat Prod Rep 35:1082-1096
Dodge, Greg J; Ronnow, Danialle; Taylor, Richard E et al. (2018) Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase. ACS Chem Biol 13:2699-2707
Skiba, Meredith A; Sikkema, Andrew P; Moss, Nathan A et al. (2017) A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit. ACS Chem Biol 12:3039-3048
Maloney, Finn P; Gerwick, Lena; Gerwick, William H et al. (2016) Anatomy of the ?-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate. Proc Natl Acad Sci U S A 113:10316-21
Stull, Frederick W; Bernard, Steffen M; Sapra, Aparna et al. (2016) Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase. Biochemistry 55:3261-9
Skiba, Meredith A; Sikkema, Andrew P; Fiers, William D et al. (2016) Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase. ACS Chem Biol 11:3319-3327

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