RUI: Biosynthesis of Diketopiperazine Natural Products from Aminoacyl-tRNAs

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.