Polyketide synthases (PKSs) are a family of structurally and mechanistically related multi-enzyme assemblies that catalyze the biosynthesis of numerous structurally complex and medicinally important natural products. Their overall catalytic cycles are comprised of a large number (typically greater than 25) of enzyme-catalyzed reactions through which simple building block molecules (usually CoA thioesters) are incrementally elaborated into structurally intricate products. Bacterial aromatic PKSs are one sub-class of PKSs. They are composed of 3-10 distinct subunits, which together synthesize a polyfunctional aromatic product. Over the past five years, the modularity of these multi-enzyme systems has been exploited by us and others for the engineered biosynthesis of numerous unnatural natural products. However, our understanding of the structural and mechanistic principles by which these remarkable enzymes assemble and catalyze multi-step transformations involving highly reactive intermediates is rudimentary at best. Towards this end, the specific goals for this proposal are: 1) Purification and reconstitution of the actinorhodin (act) and tetracenomycin (tcm) minimal PKSs; 2) Overexpression, purification, and reconstitution of representative auxiliary PKS components such as the act ketoreductase, act aromatase/cyclase (ARO/CYC), griseusin (gris) /ARO/CYC, tmc ARO/CYC, and the second ring cyclase from the act PKS; 3) Physico-chemical studies on minimal and extended PKS complexes, including kinetic analysis, detection of functionally relevant protein- protein interactions, and measurement of the subunit stoichiometries; 4) In vitro mutant complementation studies to probe the pathway for chain transfer between active sites of the act PKS; 5) Probing the molecular recognition features of aromatic PKSs using candidate unnatural substrates, including CoA and N-acetylcysteamine thioesters of primer and extender units, acyl carrier protein (ACP) and acyl-ACP analogs, as well as simplified mimics of the natural substrates of various auxiliary enzymes; 6) Probing the structural basis for limited interchangeability between the subunits of the act and tcm minimal PKS using mutagenesis as well as protein chemical approaches; 7) Dissecting chain length specificity of the minimal PKS using mutagenesis; 8) Dissecting the molecular recognition features of ARO/CYC subunits as well as their ability to influence the properties of other PKS subunits using mutagenesis and protein chemical approaches; 9) Development of sensitive assays capable of extracting short-chain intermediates from minimal aromatic PKSs; 10) (If time permits) Use of the above information for the synthesis of novel polyketides; and 11) (If time permits) Preliminary evaluation of NMR and X-ray crystallography for structural studies on these purified proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA077248-03
Application #
6173202
Study Section
Special Emphasis Panel (ZRG3-MCHA (01))
Program Officer
Fu, Yali
Project Start
1998-08-20
Project End
2002-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
3
Fiscal Year
2000
Total Cost
$231,435
Indirect Cost
Name
Stanford University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Fitzgerald, Jay T; Charkoudian, Louise K; Watts, Katharine R et al. (2013) Analysis and refactoring of the A-74528 biosynthetic pathway. J Am Chem Soc 135:3752-5
Holmes, Tracy C; May, Aaron E; Zaleta-Rivera, Kathia et al. (2012) Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor. J Am Chem Soc 134:17797-806
Wong, Fong T; Khosla, Chaitan (2012) Combinatorial biosynthesis of polyketides--a perspective. Curr Opin Chem Biol 16:117-23
Fitzgerald, Jay T; Henrich, Philipp P; O'Brien, Connor et al. (2011) In vitro and in vivo activity of frenolicin B against Plasmodium falciparum and P berghei. J Antibiot (Tokyo) 64:799-801
Szu, Ping-Hui; Govindarajan, Sridhar; Meehan, Michael J et al. (2011) Analysis of the ketosynthase-chain length factor heterodimer from the fredericamycin polyketide synthase. Chem Biol 18:1021-31
Fitzgerald, Jay T; Ridley, Christian P; Khosla, Chaitan (2011) Engineered biosynthesis of the antiparasitic agent frenolicin B and rationally designed analogs in a heterologous host. J Antibiot (Tokyo) 64:759-62
Zaleta-Rivera, Kathia; Charkoudian, Louise K; Ridley, Christian P et al. (2010) Cloning, sequencing, heterologous expression, and mechanistic analysis of A-74528 biosynthesis. J Am Chem Soc 132:9122-8
Das, Abhirup; Szu, Ping-Hui; Fitzgerald, Jay T et al. (2010) Mechanism and engineering of polyketide chain initiation in fredericamycin biosynthesis. J Am Chem Soc 132:8831-3
Charkoudian, Louise K; Fitzgerald, Jay T; Khosla, Chaitan et al. (2010) In living color: bacterial pigments as an untapped resource in the classroom and beyond. PLoS Biol 8:
Liu, Tiangang; Khosla, Chaitan (2010) Chemistry. A balancing act for Taxol precursor pathways in E. coli. Science 330:44-5

Showing the most recent 10 out of 37 publications