This project develops new tools for the identification, isolation, and characterization of polyketide synthases from producer organisms. We focus on anticancer polyketides from marine dinoflagellates (dinophyceae), a class of single-cellular marine eukaryotes that synthesize an extraordinary variety of bioactive polyketides, including okadaic acid, brevetoxin and amphidinolides. The biosynthesis of these polyketides has been difficult to characterize genetically due to the fact that the large nuclear genomes of dinoflagellates contain high gene duplication with multiple intron content. These complications are intensified by the fact that dinoflagellates often harbor bacterial endo- and exo-symbionts. We propose the application of a novel suite of protein methods for the general study of polyketide biosynthesis in dinoflagellates using a model system, the biosynthesis of amphidinolides in Amphidinium sp. The amphidinolides are a large class of macro ides, of which several molecules demonstrate promising anti-cancer activity. This suite of methods has been engineered to visualize, isolate, and manipulate polyketide synthases through manipulation of their carrier protein (CP) domains. Using these techniques synthase enzymes in Amphidinium lysates will be tagged with fluorescent or affinity tags, identified, and purified. Methods such as photocleavable reporter linkages, enzymatic 4'-phosphopantetheine cleavage, and phosphopantetheinyltransferase inhibitors will be examined as a means to provide pure synthases in their natural apo- and holo- state. A variety of downstream assays will be conducted on the purified synthases to identify the individual loading domains and modular organization. The methodologies introduced here constitute a new platform of proteomic tools to investigate natural product biosynthetic enzymes from modular synthases, including polyketide and non-ribosomal peptide synthases and their hybrids. These tools will allow further understanding of natural product pathways and guide metabolic engineering to produce therapeutically important molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075797-02
Application #
7125524
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (52))
Program Officer
Schwab, John M
Project Start
2005-09-23
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
2
Fiscal Year
2006
Total Cost
$321,165
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Worthington, Andrew S; Hur, Gene H; Burkart, Michael D (2011) Activity-guided engineering of natural product carrier proteins. Mol Biosyst 7:365-70
Meier, Jordan L; Patel, Anand D; Niessen, Sherry et al. (2011) Practical 4'-phosphopantetheine active site discovery from proteomic samples. J Proteome Res 10:320-9
Haushalter, Robert W; Filipp, Fabian V; Ko, Kwang-Seuk et al. (2011) Binding and ""pKa"" modulation of a polycyclic substrate analogue in a type II polyketide acyl carrier protein. ACS Chem Biol 6:413-8
Worthington, Andrew S; Porter, Douglas F; Burkart, Michael D (2010) Mechanism-based crosslinking as a gauge for functional interaction of modular synthases. Org Biomol Chem 8:1769-72
Foley, Timothy L; Yasgar, Adam; Garcia, Christopher J et al. (2010) Preparation of FRET reporters to support chemical probe development. Org Biomol Chem 8:4601-6
Meier, Jordan L; Haushalter, Robert W; Burkart, Michael D (2010) A mechanism based protein crosslinker for acyl carrier protein dehydratases. Bioorg Med Chem Lett 20:4936-9
Meier, Jordan L; Burkart, Michael D (2009) The chemical biology of modular biosynthetic enzymes. Chem Soc Rev 38:2012-45
Mercer, Andrew C; Meier, Jordan L; Torpey, Justin W et al. (2009) In vivo modification of native carrier protein domains. Chembiochem 10:1091-100
Meier, Jordan L; Niessen, Sherry; Hoover, Heather S et al. (2009) An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis. ACS Chem Biol 4:948-57
Foley, Timothy L; Young, Brian S; Burkart, Michael D (2009) Phosphopantetheinyl transferase inhibition and secondary metabolism. FEBS J 276:7134-45

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