Synthesis of aminoacyl-tRNA analogues to explore non-ribosomal peptide synthesis in bacteria – MimictRNA
Synthesis of aminoacyl-tRNA analogues to explore non-ribosomal peptide synthesis in bacteria
Our project will provide for the first time insight into the catalytic mechanism of Fem transferases, structural data on tRNA recognition by these enzymes, and insight into the mechanisms that control the flux of aminoacyl-tRNAs into protein and peptidoglycan synthesis. The project should bring all the tools required for the design of drugable inhibitors of Fem transferases and other tRNA- dependent bacterial targets, such as the aminoacyl-phosphatidylglycerol synthetases.<br />
Synthesis of aminoacyl-tRNA analogues to explore non-ribosomal peptide synthesis in bacteria
The major focus of the present proposal concerns the catalytic mechanism of FemX, a representative of the tRNA-dependent transferases involved in peptidoglycan synthesis. The first substrate of this enzyme, UDP-MurNAc-pentapeptide (UM5K) is a composite structure including a carbohydrate, a nucleotide and a peptide with unusual amide links. The chemical synthesis of this molecule remains particularly challenging and it is unlikely that versatile routes to analogues can be developed. For this reason, we propose to develop enzymatic synthesis of this molecule and access to modifications of its structure via protein engineering. The second substrate Ala-tRNAAla is even more complex. We have developed semi-synthesis of aminoacylated tRNAs and analogues based on organic synthesis of modified dinucleotides and their enzymatic ligation to RNA molecules obtained by in vitro transcription or by the classical solid support synthesis. Our first objective is the synthesis of functionalized analogues of the Ala-tRNA and UM5K substrates that will get access to bi-substrates by the Huisgen-Sharpless cycloaddition reaction. The Cu(I)- and FemX-catalyzed reaction will afford high affinity inhibitors that will be use to determine the structure of complexes and understand the interaction of the enzyme with the tRNA. The second objective is to explore the role of FemX in aminoacyl isomerization between the O2’ and O3’ hydroxyls of the tRNA. We will synthesize phospho-derivatives of the tRNA that will mimic the tetrahedral intermediary of the transacylation reaction and trap a relevant conformation of FemX for crystallogenesis.
The main objectives of the proposal was to investigate the catalytic mechanism of FemXWv using bi-substrates containing tRNA covalently linked to peptidoglycan precursor analogues. In order to synthesize the bi-substrates analogues, we introduce azides and alkynes into the tRNAs and UM5K, respectively. Cu(I)-catalyzed cycloaddition afford the desired bi-substrate. Azide-tRNA analogues was obtained by two strategies. The first strategy involves an enzymatic ligation by T4 RNA ligase and the second strategy is based on the solid phase synthesis. The solid-phase synthesis methods (developed in collaboration with Prof. Tom Brown, Oxford University) lead to short RNA micro-helixes containing matching base-pairs. Five 2’-azido RNA duplexes, which contain 2 to 7 base pairs have been synthezised and used to obtained peptidyl-RNA conjugates. Partner 3 synthesized UDP-MurNAc-pentapeptide and lipid II compounds in both unlabeled and radiolabeled forms. We also successfully synthesized several analogues of these two natural compounds: meso-cystine-containing UDP-MurNAc-pentapeptide and UDP-MurNAc-pentadepsipeptide, alkyne-lipid II and lipid II (-pentadepsipeptide). All synthesized compounds were checked by MALDI-TOF mass spectrometry and amino acid / amino sugar analyses.
The peptidyl-RNA conjugates were found to inhibit the non-ribosomal FemXWv aminoacyl-transferase with IC50 of 2 nM to 930 nMol and one of them was used to successfully obtain the crystallographic structure of FemXWv in complex with the Peptidyl-RNA. We have also obtained interesting results in the hemi-synthesis of 3’ fluoro-analogues of Ala-tRNAAla. The presence of fluorine in position 3’ blocks Ala at position 2’ by preventing spontaneous migration of the residue between positions 2’ and 3’. NMR analyses showed that substitution of the 3’ hydroxyl group by fluorine in the ribo configuration favors the S-type conformation of the furanose ring of terminal adenosine A76. In contrast, the N-type conformation is favored by the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. Our compounds will provide insight into substrate recognition by Fem transferase and the Ala-tRNA synthetase.
We have investigated the formation of the triazole unit to link the tRNAAla analogs and alkyne-peptidoglycan, within the active site of FemXWv in the absence of Cu(I). By these approaches, (the in situ reaction), we tried to obtain molecules suitable for co-crystallization with FemXWv. Despite numerous attempts, FemXWv-catalyzed cycloaddition has not yet been observed. Other substrates analogues and other Fem enzymes will be used to achieve this objective.
1. “Synthesis of 3’-Fluoro-tRNA Analogues to explore non-ribosomal peptide synthesis in bacteria”, L. Iannazzo, G. Laisné, M. Fonvielle, E. Braud, J-P. Herbeuval, M. Arthur, M. Etheve-Quelquejeu, Chembiochem, 2015, 16, 477-486.
2. «Synthesis of 3'-triazoyl-dinucleotides as precursors of stable Phe-tRNAPhe and Leu-tRNALeu analogues«, M. Santarem, M. Fonvielle, N. Sakkas, G. Laisné, M. Chemama, J-P. Herbeuval, E. Braud, M. Arthur, M. Etheve-Quelquejeu, Bioorg. Med. Chem. Lett., 2014, 24, 3231-3233.
1 chapitre : “Covalent functionalization of nucleic acids” M. Arthur & M. Etheve-quelquejeu, In Chemistry of Organo-Hybrids: Synthesis and Characterization of Functional Nano-Objects; Lacôte, E.; Charleux, B.; Copéret, C.; Eds.; Wiley-VCH, 2015.
In addition to their role in protein synthesis by the ribosomes, aminoacyl-tRNAs participate in various metabolic pathways as a source of ester-activated amino acids. Among the tRNA-dependent aminoacyl transferases, enzymes of the Fem family catalyze an essential step of peptidoglycan synthesis in pathogenic bacteria and are considered as attractive targets for the development of novel antibiotics. FemX, the model enzyme of the family, transfers L-Ala from Ala-tRNA to the epsilon-amino group of L-Lys in the peptidoglycan precursor UDP-MurNAc-pentapeptide (UM5K). The crystal structures of the apo-enzyme and of a UM5K-FemX complex have been determined but co-crystallization with Ala-tRNA has not been obtained. We propose to develop the semi-synthesis of highly modified aminoacyl-tRNAs and bi-substrates to explore the catalytic mechanism of FemX. We will synthesize chemical probes that will specifically interact with FemX and its substrates. Azides and alkynes will be introduced into the tRNA and in UM5K, respectively. The Huisgen-Sharpless Cu(I)-catalyzed cycloaddition reaction will afford bi-substrates containing the tRNA covalently linked to the peptidoglycan precursor. In parallel, the active center of FemX will be used to catalyze the same reaction. By this approach, we will obtain molecules suitable for co-crystallization with FemX. Because the in situ generated reaction products are likely to trap a single conformational state of FemX corresponding to the catalytically active form of the enzyme, this approach is likely to be more powerful than the conventional crystallogenesis screens made with the substrates or products of the reaction. Phospho-derivatives of the tRNA will be synthesized to mimic the putative tetrahedral intermediate resulting from the intramolecular nucleophilic attack of the carbonyl of Ala-tRNA by the vicinal ribose hydroxyl. These phospho-derivatives will also be used to trap a relevant conformation of the enzyme that allows the trans-acylation reaction of the amino acid between the 2’ and 3’ positions of Ala-tRNA to occur within the active site. The enzyme-catalyzed cycloaddition reaction will be further investigated both to identify inhibitors of FemX and to decipher the mechanism of the enzyme-assisted catalyzed cycloaddition reaction, which is poorly understood. We will assess and compare the contributions of substrate binding and substrate activation (i) in the CuI- and FemX-catalyzed cycloaddition reactions using the functionalized substrates, and (ii) in the amino acid transfer reaction catalyzed by FemX with the “natural” substrates. The information gathered on the catalytic mechanism of FemX and on the structure of its active site should provide the critical information for the rationale design of drugs active on Fem transferases from pathogenic bacteria such as methicillin-resistant staphylococci. The approach will be of broad application in RNA biology.
Project coordination
Mélanie Etheve-Quelquejeu (Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologiques)
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.
Partnership
Universite Paris Descartes Laboratoire de Chimie et de Biochimie pharmacologiques et toxicologiques
INSERM, UMR_S 872 LRMA, Equipe 12, Centre de Recherche des Cordeliers
PSUD/IBBMC Université Paris Sud/Institut de Biochimie et Biophysique Moléculaire et Cellulaire
IBBMC, UMR 8619, CNRS Institut de biochimie et Biophysique Moléculaire et Cellulaire
Help of the ANR 444,974 euros
Beginning and duration of the scientific project:
September 2013
- 42 Months
