This program investigates the role of protein-protein interactions in natural product modular synthases, a group of biosynthetic enzymes of three types: polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and fatty acid synthase (FAS). Many of these natural products serve as anti-cancer agents or antibiotics, while others are pathogenic toxins. Recent evidence indicates that protein-protein interactions between the carrier protein domain and catalytic domains of these pathways are key to proper catalysis and processivity. These critical interactions are transient in nature and remain largely uncharacterized. We have developed tools that allow synthetic modification of carrier protein domains, and here we leverage these tools to increase the lifetime of these protein-protein interactions using substrate mimics and covalent cross-linking inhibitors. These tools will allow us to visualize the molecular details by which carrier proteins bind tethered substrates prior to catalysis and how they interact with cognate catalytic domains. Through these studies, we intend to gain a more complete understanding of the dynamics of these catalytic events through analysis of solution phase NMR spectra. NMR perturbation comparisons of modified carrier proteins and titration experiments in the presence of catalytic domains will offer us an ability to accurately pinpoint the residues involved in substrate sequestration and protein-protein interaction. We will further validate our findings through site-directed mutagenesis, hydrogen/deuterium exchange NMR, and isothermal titration calorimetry. A deeper understanding of these protein-protein interactions will affect emerging fields in drug discovery. Using these tools to guide new interactions will enable combinatorial biosynthesis of new pharmacophores, while new drug targets may be discovered through abrogating these interactions in pathogen biosynthesis.

Public Health Relevance

This program investigates the activity of modular synthases, a group of natural product biosynthetic enzymes of three types: polyketide synthase, non-ribosomal peptide synthase, and fatty acid synthase. These three subgroups are responsible for the production of antibiotics that represent major constituents of medicinal therapeutics, toxins that support pathogenesis for infection, and primary metabolites essential for life. These studies will benefit human health by enabling manipulation of these pathways to provide new therapeutic drugs and to identify new targets for drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095970-03
Application #
8623136
Study Section
Special Emphasis Panel (ZRG1-SBCB-H (02))
Program Officer
Gerratana, Barbara
Project Start
2012-03-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
3
Fiscal Year
2014
Total Cost
$265,050
Indirect Cost
$94,050
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
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
Moynié, Lucile; Hope, Anthony G; Finzel, Kara et al. (2016) A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA. J Mol Biol 428:108-20
McCulloch, Ian P; La Clair, James J; Jaremko, Matt J et al. (2016) Fluorescent Mechanism-Based Probe for Aerobic Flavin-Dependent Enzyme Activity. Chembiochem 17:1598-601
Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G et al. (2016) Trapping of the Enoyl-Acyl Carrier Protein Reductase-Acyl Carrier Protein Interaction. J Am Chem Soc 138:3962-5
Vickery, Christopher R; La Clair, James J; Burkart, Michael D et al. (2016) Harvesting the biosynthetic machineries that cultivate a variety of indispensable plant natural products. Curr Opin Chem Biol 31:66-73
Rivera Jr, Heriberto; Dhar, Sachin; La Clair, James J et al. (2016) An unusual intramolecular trans-amidation. Tetrahedron 72:3605-3608
Jaremko, Matt J; Lee, D John; Opella, Stanley J et al. (2015) Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL. J Am Chem Soc 137:11546-9
Konno, Sho; Ishikawa, Fumihiro; Suzuki, Takehiro et al. (2015) Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases. Chem Commun (Camb) 51:2262-5
Lindert, Steffen; Tallorin, Lorillee; Nguyen, Quynh G et al. (2015) In silico screening for Plasmodium falciparum enoyl-ACP reductase inhibitors. J Comput Aided Mol Des 29:79-87
Beld, Joris; Lee, D John; Burkart, Michael D (2015) Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering. Mol Biosyst 11:38-59

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