Matthieu Fonvielle

Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemXWv is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemXWv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1–C72 and G2–C71 base pairs of the acceptor stem as critical for FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues.

Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemXWv. Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemXWv with the acceptor arm of tRNAGly and with l-Ser or larger residues quantitatively accounts for the preferential transfer of l-Ala observed with complete aminoacyl-tRNAs. The main FemXWv identity determinant was identified as the penultimate base pair (G2-C71) of the acceptor arm instead of G3•U70 for the alanyl-tRNA synthetase. FemXWv tolerated a configuration inversion of the Cα of l-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemXWv is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNAGly from the active site.

Decoding the Logic of the tRNA Regiospecificity of Nonribosomal FemXWv Aminoacyl Transferase

Aminoacyl-tRNAs are key intermediates in protein synthesis. They act as adapters between the codons of mRNA and the growing polypeptide chain in the ribosome. The vicinal hydroxy groups at the 2’and 3’-positions of the terminal nucleotide (A) of tRNA have pivotal roles in the function of these molecules. The tRNA molecules are esterified by aminoacyl-tRNA synthetases, which catalyze the transfer of a specific aminoacyl residue from an adenylate to the 2’or 3’-hydroxy group of A (Scheme 1). Transesterification between the 2’and 3’-positions occurs in the absence of an enzyme with a rate and thermodynamic equilibrium of the order of 5 s 1 and 1, respectively. The A site of the ribosome is specific for the 3’-O-aminoacyl isomer, and the 3’ linkage to the tRNA is conserved in the product of the peptidyl-transfer reaction. The 2’-hydroxy group of the peptidyl-tRNA is thought to assist catalysis of this reaction. Besides their role in protein synthesis, aminoacyl-tRNAs participate in various metabolic pathways, such as the synthesis of cyclodipeptides or the aminoacylation of proteins and membrane phosphatidylglycerol. Transferases of the Fem family catalyze the incorporation of amino acids into peptidoglycan precursors to form a side chain that contains the amino group used as an acyl acceptor in the final cross-linking step of cell-wall synthesis (Scheme 1). The specificity of these enzymes is essential for bacteria, since misincorporated amino acids can act as chain terminators and block peptidoglycan polymerization. Because of their key role in peptidoglycan metabolism, Fem transferases are considered attractive targets for the development of novel antibiotics. We previously used chemical acylation of RNA helices with natural and nonproteinogenic amino acids to gain insight into the specificity of FemXWv of Weissella viridescens, [12, 13] a model enzyme of the Fem family. A combination of modifications in the RNA and aminoacyl moieties of the substrate revealed that unfavorable interactions of FemXWv with the acceptor arm of tRNA and with l-Ser or larger residues quantitatively account for the preferential transfer of l-Ala observed with complete aminoacyl-tRNAs. 13] The main FemXWv identity determinant of Ala-tRNA Ala was found to be the penultimate base pair, G–C, which is replaced with C–G in tRNA isoacceptors. 13] In this study, we synthesized nonisomerizable mimics of Ala-tRNA that contained 2’-deoxyadenosine or 3’-deoxyadenosine to lock the amino acid in the 3’and 2’-position, respectively (Scheme 2). We also synthesized nonisomerizable aminoacyl-tRNA analogues by replacing the ester bond connecting the amino acid residue to the terminal nucleotide with a triazole ring (Scheme 3). We synthesized these molecules to determine the regiospecificity of FemXWv for the 3’ and 2’ isomers and to evaluate the role of the adjacent hydroxy group in the transfer reaction. Ala-tRNA analogues containing a terminal 2’or 3’deoxyadenosine residue and a 24 nucleotide (nt) helix mimicking the acceptor arm of the tRNA (Figure 1) were obtained by semisynthesis (see the Supporting Information and Scheme 2) and assayed as substrates of FemXWv

Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNAPhe is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNALeu isoacceptors as a second substrate. We show that the G1-C72 pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6–α7 or D205 located in the loop β6–α8 affected Leu-tRNALeu isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites.

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by β-Lactamase BlaC.

Combinations of β-lactams of the carbapenem class, such as meropenem, with clavulanate, a β-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual β-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum β-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the β-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable β-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.

Synthesis of 3′-Fluoro-tRNA Analogues for Exploring Non-ribosomal Peptide Synthesis in Bacteria

Aminoacyl‐tRNAs (aa‐tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl‐tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi‐synthesis of 3′‐fluoro analogues of Ala‐tRNAAla. The presence of fluorine in the 3′‐position blocks Ala at the 2′‐position by preventing spontaneous migration of the residue between positions 2′ and 3′. NMR analyses showed that substitution of the 3′‐hydroxy group by fluorine in the ribo configuration favours the S‐type conformation of the furanose ring of terminal adenosine A76. In contrast, the N‐type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala‐tRNA synthetases.

Synthesis of Avibactam Derivatives and Activity on β-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 μg mL-1 for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.