Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This renewal application of a highly productive collaborative program proposes to employ the versatile and well-characterized Streptomyces Venezuela pikromycin PKS, as well as the erythromycin, tylosin, curacin and bryostatin pathways which were the subjects of expanded detailed analysis during the previous cycle of support and are now poised for major new progress. These systems each bear fascinating biochemical features that will expand our understanding of the specificity and structural characteristics that lead to biological activity within and between natie and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, keto group processing, and termination that lea to the remarkable chemical diversity of polyketide natural products. Detailed biochemical analysis, along with X-ray and cryoEM structural biology, and molecular dynamics approaches will be applied to probe substrate specificity. Moreover, synthetic chemistry of natural and near-natural substrates will be employed to develop chemoenzymatic approaches to enable pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents.
Specific aims i nclude: I. Molecular analysis of bacterial modular polyketide synthases. We will design and employ natural and unnatural synthetic substrates and extender units to explore selectivity and tolerance in chain loading, elongation and processing in the terminal modules of Pik (modules 5 and 6), DEBS (modules 5 and 6), Tyl (modules 6 and 7), and select Cur PKS modules. II. Develop mutational strategies to engineer modular PKSs with greater catalytic efficiency toward unnatural substrates. A high-throughput bioactivity-based screen will be developed to assess the efficiency of mutant PKS modules for improved activity toward target unnatural substrates. III. Molecular analysis of bacterial symbiont trans-AT modular PKSs and ?ranching. We will explore the protein recognition determinants for trans-AT interactions, substrate selectivity, and structure and function using synthetic substrates, biochemical analysis, x-ray crystallography, cryoEM, and FT-ICR MS. In addition, a proof-of-concept method will be developed to interrogate biochemical function using bryostatin (Bry) PKS modules 3 and 4 and BryP/surrogate trans-ATs and ?ranching enzymes.

Public Health Relevance

The proposed research will focus on elucidating the detailed function of complex biosynthetic machines that create chemically diverse, biologically active natural products. The ability to understand and subsequently engineer these remarkable biochemical systems will create new opportunities to discover and develop effective drugs for the treatment of human diseases, including cancer, infectious diseases, and Alzheimer's.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM076477-09
Application #
8815375
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Gerratana, Barbara
Project Start
2006-01-10
Project End
2019-04-30
Budget Start
2015-08-15
Budget End
2016-04-30
Support Year
9
Fiscal Year
2015
Total Cost
$386,249
Indirect Cost
$126,249
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Slocum, Samuel T; Lowell, Andrew N; Tripathi, Ashootosh N et al. (2018) Chemoenzymatic Dissection of Polyketide ?-Branching in the Bryostatin Pathway. Methods Enzymol 604:207-236
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
Lowell, Andrew N; DeMars 2nd, Matthew D; Slocum, Samuel T et al. (2017) Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization. J Am Chem Soc 139:7913-7920
Hansen, Douglas A; Koch, Aaron A; Sherman, David H (2017) Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides. J Am Chem Soc 139:13450-13455
Koryakina, Irina; Kasey, Christian; McArthur, John B et al. (2017) Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering. ACS Chem Biol 12:114-123
Koch, Aaron A; Hansen, Douglas A; Shende, Vikram V et al. (2017) A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst. J Am Chem Soc 139:13456-13465
Tripathi, Ashootosh; Choi, Si-Sun; Sherman, David H et al. (2016) Thioesterase domain swapping of a linear polyketide tautomycetin with a macrocyclic polyketide pikromycin in Streptomyces sp. CK4412. J Ind Microbiol Biotechnol 43:1189-93
DeMars 2nd, Matthew D; Sheng, Fang; Park, Sung Ryeol et al. (2016) Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway. ACS Chem Biol 11:2642-54
Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon et al. (2015) Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides. Proc Natl Acad Sci U S A 112:12956-61
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

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