RESUMO
Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic ß2 and ß7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.
Assuntos
Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderiaceae/efeitos dos fármacos , Burkholderiaceae/genética , Cromatografia em Gel , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.
Assuntos
Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ensaios Enzimáticos , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Cyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. At present, there are no structural details of the CDPS:tRNA interaction available. Using AlbC, a CDPS that produces cyclo(l-Phe-l-Phe), the interaction between AlbC and its Phe-tRNA substrate was investigated. Simulations of models of the AlbC:tRNA complex, proposed by rigid-body docking or homology modeling, demonstrated that interactions with residues of an AlbC α-helix, α4, significantly contribute to the free energy of binding of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well the available biochemical data, and the results of in vivo assay experiments performed in this work and guided by simulations. In molecular dynamics simulations, the phenylalanyl group predominantly occupied the two positions observed in the experimental structure of AlbC in the dipeptide intermediate state, suggesting that tRNAs of the first and second substrates interact with AlbC in a similar manner. Overall, given the high degree of sequence and structural similarity among the members of the CDPS NYH protein subfamily, the mechanism of the protein:tRNA interaction is expected to be pertinent to a wide range of proteins interacting with tRNA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Modelos Moleculares , Mutação , Peptídeo Sintases/genética , Peptídeos Cíclicos/química , Conformação Proteica em alfa-Hélice , Aminoacil-RNA de Transferência/químicaRESUMO
Covering: Up to mid-2019 Cyclodipeptide synthases (CDPSs) catalyse the formation of cyclodipeptides using aminoacylated-tRNA as substrates. The recent characterization of large sets of CDPSs has revealed that they can produce highly diverse products, and therefore have great potential for use in the production of different 2,5-diketopiperazines (2,5-DKPs). Sequence similarity networks (SSNs) are presented as a new, efficient way of classifying CDPSs by specificity and identifying new CDPS likely to display novel specificities. Several strategies for further increasing the diversity accessible with these enzymes are discussed here, including the incorporation of non-canonical amino acids by CDPSs and use of the remarkable diversity of 2,5-DKP-tailoring enzymes discovered in recent years.
Assuntos
Biotecnologia/métodos , Dicetopiperazinas/síntese química , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Engenharia de Proteínas/métodos , Aminoácidos/química , Dicetopiperazinas/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cß dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. RESULTS: We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. CONCLUSIONS: Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.
Assuntos
Biotecnologia/métodos , Dicetopiperazinas/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Peptídeo Sintases/metabolismo , Vias Biossintéticas/genética , Dicetopiperazinas/química , Escherichia coli/genética , Oxirredutases/genética , Peptídeo Sintases/genética , FilogeniaRESUMO
Aminoacyl-tRNAs were long thought to be involved solely in ribosome-dependent protein synthesis and essential primary metabolism processes, such as targeted protein degradation and peptidoglycan synthesis. About 10 years ago, an aminoacyl-tRNA-dependent enzyme involved in the biosynthesis of the antibiotic valanimycin was discovered in a Streptomyces strain. Far from being an isolated case, this discovery has been followed by the description of an increasing number of aminoacyl-tRNA-dependent enzymes involved in secondary metabolism. This review describes the three groups of aminoacyl-tRNA-dependent enzymes involved in the synthesis of natural products. Each group is characterized by a particular chemical reaction, and its members are predicted to share a specific fold. The three groups are cyclodipeptide synthases involved in diketopiperazine synthesis, LanB-like dehydratases involved in the posttranslational modification of ribosomal peptides, and transferases from various biosynthesis pathways.
Assuntos
Produtos Biológicos/metabolismo , Enzimas/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Catálise , Enzimas/química , Conformação Proteica , Especificidade por SubstratoRESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
Assuntos
Peptídeo Sintases/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Relação Estrutura-AtividadeRESUMO
The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5-diketopiperazines (2,5-DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an inâ vivo method for incorporating non-canonical amino acids (ncAAs) into 2,5-DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5-DKP biosynthetic pathways. CDPSs use aminoacyl-tRNAs as substrates. We exploited the natural ability of aminoacyl-tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non-canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5-DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide-tailoring enzymes found in 2,5-DKP biosynthetic pathways.
Assuntos
Aminoácidos/metabolismo , Dicetopiperazinas/metabolismo , Peptídeo Sintases/metabolismo , Aminoácidos/química , Dicetopiperazinas/química , Conformação MolecularRESUMO
Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.
Assuntos
Proteínas de Bactérias/química , Dipeptídeos/química , Proteínas Fúngicas/química , Peptídeo Sintases/química , Peptídeos Cíclicos/química , Motivos de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biologia Computacional , Ciclização , Bases de Dados Genéticas , Dipeptídeos/biossíntese , Dipeptídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Expressão Gênica , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Filogenia , Estrutura Terciária de Proteína , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
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
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
BACKGROUND: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. RESULTS: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. CONCLUSIONS: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.
Assuntos
Escherichia coli/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Citoplasma/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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
We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities, focusing on the biosynthetic pathways involving cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. Distinct from nonribosomal peptide synthetases (NRPSs), the other family of enzymes synthesizing DKPs, CDPSs bridge the primary and secondary metabolic pathways by hijacking aminoacyl-tRNAs to produce DKPs. This review includes a comprehensive description of the state of the art for CDPS-dependent pathways, and highlights the ways in which this knowledge could be used to increase the diversity of natural DKPs by pathway engineering.
Assuntos
Produtos Biológicos/síntese química , Dicetopiperazinas/síntese química , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Sequência de Aminoácidos , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Dicetopiperazinas/química , Dicetopiperazinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Conformação ProteicaRESUMO
The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 A), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 A apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Dipeptídeos/química , Mycobacterium tuberculosis/enzimologia , Peptídeos Cíclicos/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ligação de Hidrogênio , Mycobacterium tuberculosis/genética , Ressonância Magnética Nuclear Biomolecular , Oxigênio/química , Oxigênio/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).
Assuntos
Peptídeo Sintases/metabolismo , RNA de Transferência/metabolismo , Biocatálise , Dados de Sequência Molecular , Peptídeo Sintases/química , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2-26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.
Assuntos
Dicetopiperazinas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , PrenilaçãoRESUMO
The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.
Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Genes Bacterianos/genética , Família Multigênica , Streptomyces/genética , Antibacterianos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Dicetopiperazinas/química , Hidroxilação , Modelos Químicos , Estrutura Molecular , Mutação , Streptomyces/metabolismoRESUMO
Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs.