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Flexizymes help undestanding the functioning of cyclodipeptide synthases

​A team from I2BC (SBIGeM), in collaboration with DMTS, took advantage of flexizymes technology to elucidate the functioning of the synthases of cyclodipeptides, enzymes implicated in the synthesis of 2,5-diketopiperazines, compounds that represent an unequaled source of bioactive molecules for the pharmacopoeia.

Published on 5 November 2020

​2,5-Diketopiperazines (2,5-DKP), produced by fungi, bacteria, plants and animals, possess remarkable biological properties (antibacterial, antiviral, anticancer or anti-inflammatory) which make them very attractive for pharmacopoeia. Muriel Gondry's team (I2BC/SBIGeM) has been studying the bacterial biosynthetic pathways of these molecules for several years. 2,5-DKP are synthesized in several steps: firstly, the cyclodipeptide synthases (CDPS), discovered in 2009 by the team, produce cyclodipeptides, using the amino acids loaded on the transfer RNAs (AA-tRNA). These cyclodipeptides are then modified by other enzymes to form 2,5-DKP.

Despite a low level of sequence identity and the existence of two subfamilies of CDPS, these enzymes all appear to use a similar mechanism, relying on the successive binding of two AA-tRNA substrates and the formation of successive aminoacyl- enzyme and dipeptidyl-enzyme intermediates. For each of the two AA-tRNA substrates, the region of the tRNA recognized by CDPS is unknown, although some studies suggest that this is the acceptor arm. This hypothesis was tested in the present study.

For this purpose, researchers took advantage of flexizymes technology, developed by a Japanese team. Flexizymes are ribozymes (RNA possessing enzyme-like activity) capable of aminoacylating a wide variety of RNAs. Using this technology, implemented and adapted in the laboratory, the researchers produced and purified various shortened AA-tRNAs which were tested in vitro for their ability to be substrates for CDPS. They thus observed that molecules mimicking the acceptor arm of tRNAs (AA-microHx) (figure) are substrates as effective as AA-tRNAs, demonstrating that the acceptor arm of tRNAs is the only region necessary for the activity of CDPS.

 A: left, schema of a tRNA with the acceptor arm shown in green; right, schema of the shortened tRNA mimicking the tRNA acceptor arm (microHx). B: microHx is aminoacylated by flexizymes to form AA-microHX, which the present study demonstrates to be a substrate of CDPS. © N.Canu/CEA

The method developed in this study could be used for the mechanistic study of other enzymes using AA-tRNA as substrate. It should also allow the incorporation into cyclopeptides of unnatural analogues of amino acids via the synthesis by flexizymes of tRNA or micro-Hx loaded with these analogues which will then be tested as substrates of CDPS.

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