RESUMO
Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.
Assuntos
Metiltransferases , RNA , Adenosina , Domínio Catalítico , Metilação , Metiltransferases/metabolismo , RNA/metabolismoRESUMO
Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1',5'-anhydrohexitol (HNA), 2'-fluoro ribose (2'F-RNA) and 2'-fluoro arabinose. L-Ala-tXNA containing HNA or 2'F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.
Assuntos
Aminoácidos , RNA de Transferência de Alanina , Ácidos Nucleicos/química , Oligonucleotídeos/química , Peptídeos , RNA de Transferência de Alanina/químicaRESUMO
The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tuâ¢GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tuâ¢GTP and the ribosome were previously identified. Here, we show that these 'non-proteogenic' tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tuâ¢GTP competitively inhibited FmhB by sequestration of 'proteogenic' aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tuâ¢GTP and limited recognition of proteogenic tRNAs by FmhB.
Assuntos
Peptidoglicano/biossíntese , RNA Bacteriano/metabolismo , RNA de Transferência de Glicina/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Ligação Competitiva , Parede Celular/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator Tu de Elongação de Peptídeos/metabolismo , Ligação ProteicaRESUMO
The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.
Assuntos
Carbapenêmicos/química , Antibacterianos/farmacologia , Parede Celular , Meropeném , Peptidoglicano , Peptidil TransferasesRESUMO
ß-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of ß-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of ß-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.
Assuntos
Peptidil Transferases , beta-Lactamas , Antibacterianos , Carbapenêmicos , Química Click , Proteínas de Ligação às Penicilinas , PeptidoglicanoRESUMO
The Enterococcus faecium l,d-transpeptidase (Ldtfm) mediates resistance to most ß-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by ß-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding (KD [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various ß-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the ß-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of ß-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the ß-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.
Assuntos
Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Cisteína/química , Enterococcus faecium/enzimologia , Peptidil Transferases/metabolismo , Acilação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Domínio Catalítico/fisiologia , Enterococcus faecium/metabolismo , Metilação , Peptidoglicano/químicaRESUMO
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.
Assuntos
Enterococcus faecium/enzimologia , Mycobacterium tuberculosis/enzimologia , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Transaminases/metabolismo , Parede Celular/metabolismo , Enterococcus faecium/química , Enterococcus faecium/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Peptidil Transferases/efeitos dos fármacos , beta-Lactamas/químicaRESUMO
Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNAGly and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 =56â nm). The synthetic route was exploited to generate substrates and inhibitors containing d-lactate residue (d-Lac) instead of d-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.
Assuntos
Carboidratos/química , Parede Celular/enzimologia , Inibidores Enzimáticos/química , Lipídeos/química , RNA/química , Técnicas de Síntese em Fase Sólida/métodos , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Carboidratos/síntese química , Carboidratos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Descoberta de Drogas , Farmacorresistência Bacteriana , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Lipídeos/síntese química , Lipídeos/farmacologia , Peptidoglicano/metabolismo , RNA/síntese química , RNA/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Especificidade por SubstratoRESUMO
There is a renewed interest for ß-lactams for treating infections due to Mycobacterium tuberculosis and M.â abscessus because their ß-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 16â µg mL-1 for both species). Mechanistically, ß-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.
Assuntos
Compostos Azabicíclicos/síntese química , Mycobacterium tuberculosis/enzimologia , Peptidoglicano/biossíntese , Inibidores de beta-Lactamases/química , beta-Lactamases/química , Compostos Azabicíclicos/química , Mycobacterium tuberculosis/química , Peptidoglicano/química , beta-Lactamases/metabolismoRESUMO
We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.
Assuntos
Técnicas de Química Sintética/métodos , RNA de Transferência/química , Ribonucleotídeos/química , Ribose/análogos & derivados , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência/síntese química , RNA de Transferência/metabolismo , Ribonucleotídeos/síntese química , Ribonucleotídeos/metabolismo , Ribose/síntese química , Ribose/metabolismo , Weissella/enzimologia , Weissella/metabolismoRESUMO
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-tRNA(Phe) 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-tRNA(Leu) isoacceptors as a second substrate. We show that the G(1)-C(72) 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 ß6-α8 affected Leu-tRNA(Leu) 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.
Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Peptídeo Sintases/química , Aminoacil-RNA de Transferência/química , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Especificidade por SubstratoRESUMO
RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3' end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv . The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity.
Assuntos
Aminoaciltransferases/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Peptídeos/metabolismo , RNA de Transferência/metabolismo , RNA/biossíntese , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Aminoaciltransferases/química , Reagentes de Ligações Cruzadas/química , Modelos Moleculares , Conformação Molecular , Peptídeos/química , RNA/química , RNA de Transferência/química , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
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-tRNA(Ala) . 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.
Assuntos
Bioquímica/métodos , Flúor/química , RNA de Transferência de Alanina/química , Técnicas de Química Sintética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Conformação de Ácido Nucleico , RNA Ligase (ATP)/química , RNA de Transferência de Alanina/síntese química , Proteínas Virais/químicaRESUMO
Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn(85), Phe(168), and Trp(182). The propensity of the cYY tyrosyl to point toward Arg(386) was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.
Assuntos
Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Dipeptídeos/química , Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/química , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Soluções , Especificidade por SubstratoRESUMO
We report here the synthesis of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3'-azido-3'-deoxyadenosine via the Cu(I)-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA(Phe) analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.
Assuntos
Nucleotídeos/síntese química , RNA de Transferência de Leucina/química , RNA de Transferência de Fenilalanina/química , Triazóis/química , Modelos Moleculares , Conformação Molecular , Nucleotídeos/química , RNA de Transferência de Leucina/síntese química , RNA de Transferência de Fenilalanina/síntese química , Triazóis/síntese químicaRESUMO
Six genes encoding putative high molecular weight penicillin-binding proteins (Pbp) are present in the genome of the ß-lactam-resistant strain Corynebacterium jeikeium K411. In this study, we show that pbp2c, one of these six genes, is present in resistant strains of Corynebacteriaceae but absent from sensitive strains. The molecular study of the pbp2c locus from C. jeikeium and its heterologous expression in Corynebacterium glutamicum allowed us to show that Pbp2c confers high levels of ß-lactam resistance to the host and is under the control of a ß-lactam-induced regulatory system encoded by two adjacent genes, jk0410 and jk0411. The detection of this inducible resistance may require up to 48 h of incubation, particularly in Corynebacterium amycolatum. Finally, the Pbp2c-expressing strains studied were resistant to all the ß-lactam antibiotics tested, including carbapenems, ceftaroline, and ceftobiprole.
RESUMO
Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.
Assuntos
Aminoaciltransferases/metabolismo , Oligopeptídeos/química , RNA/química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Aminoaciltransferases/antagonistas & inibidores , Catálise , Cobre/química , Reação de Cicloadição , Cinética , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ligação Proteica , RNA/síntese química , RNA/metabolismo , RNA Ligase (ATP)/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Transferases de Grupos Nitrogenados/antagonistas & inibidores , RNA de Transferência de Alanina/síntese química , RNA de Transferência de Alanina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Modelos Moleculares , Conformação Molecular , Transferases de Grupos Nitrogenados/metabolismo , RNA de Transferência de Alanina/química , Relação Estrutura-AtividadeRESUMO
Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.
Assuntos
Proteínas de Bactérias/química , Dipeptídeos/biossíntese , Peptídeo Sintases/química , Peptídeos Cíclicos/biossíntese , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologiaRESUMO
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 FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.