Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity

Canu, Nicolas; Tellier, Carine; Babin, Morgan; Thaï, Robert; Ajel, Inès; Seguin, Jérôme; Cinquin, Olivier; Vinck, Robin; Moutiez, Mireille; Belin, Pascal; Cintrat, Jean‐Christophe; Gondry, Muriel

doi:10.1093/nar/gkaa903

Cited by 6 publications

(2 citation statements)

References 52 publications

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“…These minimal substrates are useful tools to investigate substrate specificity, and binding to P1/P2, while also presenting an alternative to using aa-tRNA as substrates. Previously, the use of a shortened tRNA was employed in combination with aa-tRNAs on the CDPS from Nocardia brasiliensis 36 , but the hybrid reaction conditions detailed herein use a unique combination of aa-DBE and aa-tRNA as substrates which has not previously been published.…”

Section: Resultsmentioning

confidence: 99%

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

2022

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Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.

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Section: Resultsmentioning

confidence: 99%

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

2022

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“…NRPSs use free α-amino acids as substrates for cyclic dipeptide formation. In contrast, CDPSs and RCDPSs utilize aminoacyl-tRNAs as substrates [30][31][32][33][34][35] , diverting them from their canonical role in ribosomal protein synthesis.…”

Section: Introductionmentioning

confidence: 99%

Cyclodipeptide oxidase is an enzyme filament

Andreas,

Giessen

2023

Preprint

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Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of theStreptomyces nourseienzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,β-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that all CDO-like enzymes are likely enzyme filaments. Our work represents the first structural characterization of a CDO. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

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Expanding the Scope of Ribosome‐Mediated Biosynthesis in vitro using tRNA‐Aminoacylating Ribozyme

Cho,

Jin,

Jeon

et al. 2024

Israel Journal of Chemistry

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Proteins are synthesized within ribosomes through the polymerization of amino acids (AAs). This process requires prior activation of AAs through aminoacylation that attaches them to their corresponding transfer RNAs (tRNAs). Within cells, this attachment is facilitated by aminoacyl‐tRNA synthetase, resulting in a tRNA:AA conjugate. A set of ribozymes developed to acylate tRNA with non‐canonical substrates enables this process outside the confines of living cells, thereby facilitating the synthesis of novel bio‐based products. In modern biotechnology, aminoacylating ribozymes contribute to the production of innovative bio‐based materials bearing functional non‐canonical chemical substrates (NCSs) and fill the gaps in synthesizing unique polymeric backbones, extending the scope beyond traditional peptide bonds. This review summarizes current understanding of flexizymes at the molecular level and their application in generating exceptional polymeric backbones through ribosome‐mediated synthesis in vitro.

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Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity

Cited by 6 publications

References 52 publications

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Cyclodipeptide oxidase is an enzyme filament

Expanding the Scope of Ribosome‐Mediated Biosynthesis in vitro using tRNA‐Aminoacylating Ribozyme

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