CE44 - Biochimie du Vivant

Deciphering the interactions of CycloDipeptide Synthases with their aminoacyl-tRNA substrates for generating engineered enzymes – Flex-Pep

Submission summary

Understanding the functioning of enzymes involved in natural product biosynthesis improve making informed decision regarding enzyme engineering for producing molecules with therapeutic value. Cyclodipeptide synthases (CDPSs) constitute a family of tRNA-dependent enzymes involved in the biosynthesis of diketopiperazines (DKPs), a large class of microbial natural products with noteworthy biological activities, such as antibacterial, antifungal, immunosuppressive or anti-inflammatory.
CDPSs use two aminoacyl-tRNAs (AA-tRNAs), diverted from their canonical role in ribosomal protein synthesis, to catalyse the DKP-ring formation of various cyclodipeptides. Partner 1 of this proposal recently showed that CDPSs are able to incorporate non canonical amino acids (ncAAs) into their cyclodipeptide products, leading to the enzymatic synthesis of approximately 200 non canonical DKPs. However, a large number of tested ncAA-tRNAs were not recognized as substrates by CDPSs. Investigating the molecular bases of CDPSs substrate specificity, i.e. understanding the interactions between CDPSs and their AA-tRNA substrates, is essential for generating engineered enzymes with modified specificities.
CDPSs have been extensively studied to understand how they proceed. The catalytic cycle involves the binding of the first AA-tRNA and the transfer of its aminoacyl moiety on a conserved serine to form an aminoacyl-enzyme intermediate. The second AA-tRNA interacts with the intermediate and its aminoacyl moiety is transferred to form a dipeptidyl-enzyme intermediate. The dipeptidyl moiety undergoes an intramolecular cyclisation leading to the final cyclodipeptide. However, all the biochemical and structural results acquired for several years by Partner 1 and its collaborators or other groups did not provide sufficiently accurate and usable information on substrate specificity of CDPSs, in particular as regards the molecular bases of their interaction with the tRNA moieties of the substrates.
Flex-Pep is a basic research project that aims to understand the molecular bases of the interactions between CDPSs and their two AA-tRNAs in order to engineer CDPSs able to produce new cyclodipeptides. To achieve this objective, we will work with a group of CDPSs that use two different AA-tRNAs as first and second substrate and with substrate analogues having the shortest RNA lengths, hereinafter called AA-miniRNA analogues. CDPSs and AA-miniRNA analogues should form smaller and less flexible complexes suitable for biophysical and structural studies. The AA-miniRNA analogues will be prepared using flexizymes (Fx), i.e. 45-47 nucleotide-ribozymes that are versatile RNA acylation catalysts. The partners of the proposal will use a panel of biochemical, and biophysical techniques (e.g. Kinetic assays, ITC, the new switchSense technology, …) to characterize the interactions between appropriate couples of CDPSs and AA-miniRNA analogues and select the best couples for X-ray studies, which will be combined with NMR studies when relevant. Finally, the partners will use the knowledge gained on the molecular interactions between CDPSs and their substrates to engineer CDPSs using a semi-rational approach. The engineered CDPSs will be screened for the incorporation of ncAAs with clickable functions.
The new DKPs obtained will be added to the DKP library being constituted by Partner 1, whose molecules will be tested for their pharmacological activities. Moreover, the strategy used in this project could be applied to the study of the many natural product pathways in which the use of AA-tRNAs has recently emerged.

Project coordination

Muriel GONDRY (Institut de Biologie Intégrative de la Cellule)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

I2BC Institut de Biologie Intégrative de la Cellule
JOLIOT Institut des sciences du vivant FRÉDÉRIC-JOLIOT
ICSN Institut de Chimie des Substances Naturelles
JOLIOT Institut des sciences du vivant FRÉDÉRIC-JOLIOT

Help of the ANR 555,910 euros
Beginning and duration of the scientific project: January 2020 - 48 Months

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