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Laboratory | Genomics


Enzymology and Non-Ribosomal Peptide Biosynthesis

​​​​The team studies the biological synthesis of natural products. This study covers several aspects: identification of new biosynthetic pathways in microbial genomes, deciphering of these pathways including the elucidation of the synthesized products, the functional and structural characterization of biosynthetic enzymes but also synthetic biology approaches to produce natural products derivatives.

Version Française

Published on 8 February 2019

Team Leader

Muriel GONDRY
muriel.gondry@cea.fr​  / muriel.gondry@i2bc.paris-saclay.fr  
Tél : +33 1 69 08 76 47


From left to right: P. Belin, I.B. Jacques, E. Favry, M. Gondry, J. Seguin, M. Moutiez

This team is part of I2BC (Enzymology and non ribosomal peptide biosynthesis).

Ongoing research focuses on nonribosomal synthesis of peptide natural products, and more particularly, cyclodipeptides and their complex derivatives, diketopiperazines (DKPs). DKPs constitute a large class of natural products synthesized essentially by microorganisms. Their physiological roles are not well known but some DKPs would be involved in cell to cell communication processes; as to their pharmacological activities, they are often remarkable and various such as antibacterial, antiviral and anticancer (Figure 1).

Figure 1 : Some bioactive DKP. The cyclodipeptide ring is in blue.​

Identification and deciphering of new biosynthesis pathways of small peptide molecules​

Many bioactive peptides are ribosome-independently synthesized by dedicated enzymes. The majority of nonribosomal peptides are produced by large multimodular enzymes, NRPSs (NonRibosomal Peptide Synthetases). However, the team - in collaboration with the J.-L. Pernodet group (Team MMA, Microbiology Department, I2BC) - identified a new family of enzymes, the cyclodipeptide synthases (CDPSs) that produce various cyclodipeptides. These enzymes are often associated with cyclodipeptides-tailoring enzymes in biosynthetic pathways dedicated to DKP production (Figure 2). One of the goals of our team is to continue this identification of new biosynthetic pathways and decrypt the diversity of molecules synthesized by these biosynthetic pathways.

 

Figure 2: Examples  of CDPS-dependent biosynthetic pathways  with CDPS (brown genes) and cyclodipeptide-tailoring enzymes (red genes : P450s ; blue: methyltransferases;  purple: various oxygenases) in various organisms.​

 

Functional and structural characterization of biosynthesis enzymes

The thorough characterization of CDPSs is ongoing in the laboratory. Studies have shown that these enzymes divert aminoacyl tRNAs (aa-tRNAs) from their canonical role in ribosomal protein synthesis for use them as substrates and catalyze the formation of various cyclodipeptides. We have also demonstrated significant structural similarity of CDPSs with class I aminoacyl-tRNA synthetases, which catalyze the loading of amino acids to their corresponding tRNAs to form aa-tRNAs (Figure 3). 

 

Figure 3: Superposition of the CDPS AlbC structure (green) with that of TyrRS, its catalytic domain (blue),
 its tRNA-binding domain (grey) and Tyr substrate (orange).​​


Recently, we have elucidated the catalytic mechanism used by the CDPSs, which is of ping-pong type and passes through the successive formation of two covalent intermediates, an aminoacyl-enzyme then a dipeptidyl-enzyme (Figure 4). We have also identified key determinants of the CDPS specificity and this work is ongoing in the laboratory.  We are also interested in cyclodipeptides-tailoring enzymes that are associated with CDPSs in DKP biosynthetic pathways. Therefore, we studied CYP121, a Mycobacterium tuberculosis cytochrome P450, which has been described as essential to the viability of the pathogen. Our work has led to the identification of the cyclodipeptide substrate and the product of the reaction catalyzed by CYP121, as well as the determination of its substrate and reaction specificity. Moreover, as CYP121 is a potential new therapeutic target, we develop substrates analogs as selective inhibitors of CYP121.

 

Figure 4: Global view of the mechanism used by CDPSs. tRNAs are aminoacylated by aaRS (red) and delivered to the ribosome (blue) for ribosomal protein synthesis. AA-tRNAs are diverted by CDPS (green) that use them as substrates via a ping-pong mechanism.​​


Engineering of biosynthesis pathways of non-ribosomal peptides

Engineering approaches aim to increase the diversity of molecules that can be synthesized with the CDPS-dependent biosynthetic pathways. They require improving our knowledge on specificity of CDPSs and cyclodipeptide-tailoring enzymes. This work is ongoing in the laboratory both for CDPS and tailoring enzymes. It will lead us in the short term to use various synthetic biology approaches to assess the potential of the chemical diversity accessible via such biosynthetic pathways.