The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis 'or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims:
AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the """"""""molecular ruler"""""""" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis.
AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis;
AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of """"""""unnatural"""""""" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new """"""""unnatural"""""""" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM076330-01A2
Application #
7305076
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Jones, Warren
Project Start
2007-07-01
Project End
2012-04-30
Budget Start
2007-07-01
Budget End
2008-04-30
Support Year
1
Fiscal Year
2007
Total Cost
$242,018
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Valentic, Timothy R; Jackson, David R; Brady, Sean F et al. (2016) Comprehensive Analysis of a Novel Ketoreductase for Pentangular Polyphenol Biosynthesis. ACS Chem Biol 11:3421-3430
Barajas, Jesus F; Finzel, Kara; Valentic, Timothy R et al. (2016) Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain. Angew Chem Int Ed Engl 55:13005-13009
Jackson, David R; Yu, Xia; Wang, Guojung et al. (2016) Insights into Complex Oxidation during BE-7585A Biosynthesis: Structural Determination and Analysis of the Polyketide Monooxygenase BexE. ACS Chem Biol 11:1137-47
Ray, Lauren; Valentic, Timothy R; Miyazawa, Takeshi et al. (2016) A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units. Nat Commun 7:13609
Caldara-Festin, Grace; Jackson, David R; Barajas, Jesus F et al. (2015) Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases. Proc Natl Acad Sci U S A 112:E6844-51
Bruegger, Joel; Haushalter, Robert W; Haushalter, Bob et al. (2013) Probing the selectivity and protein┬Ěprotein interactions of a nonreducing fungal polyketide synthase using mechanism-based crosslinkers. Chem Biol 20:1135-46
Javidpour, Pouya; Bruegger, Joel; Srithahan, Supawadee et al. (2013) The determinants of activity and specificity in actinorhodin type II polyketide ketoreductase. Chem Biol 20:1225-34
Lee, Ming-Yue; Ames, Brian D; Tsai, Shiou-Chuan (2012) Insight into the molecular basis of aromatic polyketide cyclization: crystal structure and in vitro characterization of WhiE-ORFVI. Biochemistry 51:3079-91
Ames, Brian D; Nguyen, Chi; Bruegger, Joel et al. (2012) Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis. Proc Natl Acad Sci U S A 109:11144-9
Javidpour, Pouya; Korman, Tyler Paz; Shakya, Gaurav et al. (2011) Structural and biochemical analyses of regio- and stereospecificities observed in a type II polyketide ketoreductase. Biochemistry 50:4638-49

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