| NSF Org: |
CHE Division Of Chemistry |
| Recipient: |
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| Initial Amendment Date: | August 16, 2017 |
| Latest Amendment Date: | August 16, 2017 |
| Award Number: | 1709655 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Pui Ho
puiho@nsf.gov (703)292-0000 CHE Division Of Chemistry MPS Directorate for Mathematical and Physical Sciences |
| Start Date: | August 15, 2017 |
| End Date: | July 31, 2021 (Estimated) |
| Total Intended Award Amount: | $294,000.00 |
| Total Awarded Amount to Date: | $294,000.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
1 UNF DR JACKSONVILLE FL US 32224-7699 (904)620-2455 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
1 UNF Drive Jacksonville FL US 32224-7699 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Chemistry of Life Processes |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.049 |
ABSTRACT
Enzymes catalyze the assembly of small molecules with diverse chemical structures and biological functions that are found in nature. Natural products can act as communication signals between organisms in nature. As such, natural products are valuable as tools for the study of biological processes and are critical in agriculture and other industries. This Research in Undergraduate Institutions (RUI) award from the Chemistry of Life Processes Program to Professor Amy L. Lane at the University of North Florida and collaborator, Rajesh Viswanathan at Case Western Reserve University funds the investigation of enzymes that catalyze the assembly of natural products known as diketopiperazines. Diketopiperazines are recognized for a variety of biological functions. This project may reveal details of the function of these enzymes and offer shorter and more sustainable routes for the synthesis of novel molecules for practical applications. Cultivation of the next generation of Science, Technology, Engineering and Mathematics professionals is integrated into this RUI award via the training of undergraduates in chemical biology and the development of an inquiry-based lab for undergraduate biochemistry courses that increase student competencies at the interface of chemistry and biology.
Aminoacyl tRNAs (aa-tRNAs) are recognized for delivering amino acids to the ribosome for protein assembly. A new role of aa-tRNAs as substrates of cyclodipeptide synthases (CDPSs) was recently established. CDPSs catalyze the assembly of bioactive 2,5-diketopiperazine (DKP) natural products from two aa-tRNAs. Few known enzyme families use aa-tRNA substrates, making CDPSs unique contributors to the chemistry of life. CDPSs are understudied relative to other common biosynthetic enzymes, and it remains unclear how CDPSs recognize specific aa-tRNAs from the pool of ~50+ different aa-tRNAs in cells. This RUI project aims to decipher molecular determinants of aa-tRNA recognition by CDPSs using two recently characterized CDPSs that yield cyclo(L-Trp-L-Trp) DKP from tryptophanyl-tRNA-Trp as experimental models. A library of unnatural aa-tRNAs featuring aminoacyl and tRNA structural variations is evaluated as CDPS substrates, to determine the relationship between aa-tRNA structure and recognition by these CDPSs and provide a single step synthetic biology platform to yield novel DKPs. Molecular models of CDPSs guide the design of site-directed mutagenesis experiments to experimentally determine CDPS residues that are responsible for recognition of aa-tRNA substrates. Results from this study may illuminate the molecular basis of aa-tRNA recognition by CDPSs, enable comparison of molecular determinants of substrate recognition between CDPSs and evolutionary relatives, and extend CDPS-catalyzed DKP chemistry. As its major broader societal impacts, this project provides tools to enable the biological construction of complex DKP natural products for practical applications, yields training opportunities for future scientists, and develop sCDPSs as model enzymes for enhancing undergraduate biochemistry course curricula.
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclic dipeptides as precursors to an array of unique and bioactive 2,5-diketopiperazine (DKP) natural products. CDPSs employ two aminoacyl-tRNAs (aa-tRNAs) as the building blocks for these cyclic dipeptides. We characterized NozA and NcdA, two CDPSs from a marine bacterium named Nocardiopsis sp. Each of these enzymes catalyze the formation of cyclo-L-Trp-L-Trp (cWW) as the cyclic dipeptide precursor of the nocardioazine DKP natural products.
The objectives of this NSF project were to use these nocardioazine pathway CDPSs as models to probe the biological assembly of DKP natural products and to provide biosynthetic access to novel DKPs. This NSF project also aimed to apply CDPSs as models for project-based classroom instruction, to increase educational opportunities for the diverse undergraduate student population of University of North Florida (UNF).
The major intellectual merits of this project were: (a) we established the relationship between aminoacyl structure and aa-tRNA recognition by NozA and NcdA CDPSs, and (b) we provided new biological synthesis routes to DKPs from unnatural amino acids. This provided >10 new cyclo-L-Trp-L-Trp derivatives constructed from unnatural amino acids, and we demonstrated these molecules as suitable precursors for the biological assembly of more complex DKPs. This work also yielded bio-inspired chemical synthesis approaches for these DKP natural products.
The major broader impacts of this project were: (a) we developed a project-based lab course that resulted in the biochemical characterization of nearly a dozen CDPSs and expanded access to research-based educational opportunities for UNF students, (b) >10 UNF undergraduates gained in-depth, hands-on research experience through conducting the experiments that yielded the major intellectual merits of this project, and (c) we generated novel DKPs that may have future practical applications in fields such as medicine and agriculture.
Together, our intellectual merits and broader impacts expand the promise of enzymatic approaches for the construction of unique natural products and highlight the potential of biosynthesis projects for training of the next generation of scientists.
Last Modified: 11/29/2021
Modified by: Amy L Lane
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