Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases.

International audienceCyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs

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

International audienceCyclodipeptide 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 ␣4 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 ␣6–␣7 or D205 located in the loop ␤6–␣8 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

An Iterative Nonribosomal Peptide Synthetase Assembles the Pyrrole-Amide Antibiotic Congocidine in Streptomyces ambofaciens

SummaryCongocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS

Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis

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-tRNAPhe 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

Les cyclodipeptides synthases, une nouvelle famille d'enzymes formant des liaisons peptidiques (mise en évidence de la famille, bases moléculaires de leur fonction, caractérisation structurale de l'AlbC, un membre de la famille)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Les cyclodipeptide synthases (vers la compréhension de leur mécanismecatalytique et des bases moléculaires de leur spécificité)

Les cyclodipeptides et leurs dérivés, les dicétopipérazines (DKP), constituent une large classe de métabolites secondaires aux activités biologiques remarquables qui sont essentiellement synthétisés par des microorganismes. Les voies de biosynthèse de certaines DKP contiennent des synthases de cyclodipeptides (CDPS), une famille d enzymes récemment identifiée. Les CDPS ont la particularité de détourner les ARNt aminoacylés de leur rôle essentiel dans la synthèse protéique ribosomale pour les utiliser comme substrats et ainsi catalyser la formation des deux liaisons peptidiques de différents cyclodipeptides. Le travail de thèse présenté dans ce manuscrit a pour objectif de caractériser la nouvelle famille des CDPS. Dans un premier temps, la caractérisation tant structurale que mécanistique de la première CDPS identifiée, AlbC de Streptomyces noursei, est présentée. Puis, les résultats obtenus avec trois autres CDPS, chacune de ces enzymes ayant des caractéristiques adéquates pour approfondir l étude de la famille des CDPS, sont décrits. Ainsi, la CDPS Ndas_1148 de Nocardiopsis dassonvillei a permis d étendre nos connaissances sur les bases moléculaires de la spécificité des CDPS. La CDPS AlbC-IMI de S. sp. IMI 351155 est un bon modèle pour analyser l interaction de chacun des deux substrats nécessaires à la formation d un cyclodipeptide. Enfin, la caractérisation de la CDPS Nvec-CDPS2 chez l animal Nematostella vectensis a permis de fournir le premier exemple d enzyme d origine animale impliquée dans la synthèse peptidique non ribosomale.Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of secondary metabolites with noteworthy biological activities that are mainly synthesized by microorganisms. The biosynthetic pathways of some DKPs contain cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. CDPSs hijack aminoacyl-tRNAs from their essential role in ribosomal protein synthesis to catalyze the formation of the two peptide bonds of various cyclodipeptides. The aim of the work presented in this thesis manuscript is to characterize the CDPS family. At first, the structural and mechanistic characterization of the first identified CDPS, AlbC of Streptomyces noursei, is presented. Then, the results obtained with three other CDPSs, each of which having suitable properties to increase our understanding of the CDPS family, are described. The CDPS Ndas_1148 of Nocardiopsis dassonvillei extends our knowledge of the molecular bases of the CDPS specificity. The CDPS AlbC-IMI of S. sp. IMI 351155 is a good model to analyze the interaction of each of the two substrates required for the formation of a cyclodipeptide. Finally, the characterization of the CDPS Nvec-CDPS2 from Nematostella vectensis provides the first example of enzymes of animal origin involved in nonribosomal peptide synthesis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Aminoacyl-tRNA-Utilizing Enzymes in Natural Product Biosynthesis

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

Cyclodipeptide synthases: a promising biotechnological tool for the synthesis of diverse 2,5-diketopiperazines

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Cyclization Reaction Catalyzed by Cyclodipeptide Synthases Relies on a Conserved Tyrosine Residue

International audienceCyclodipeptide 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. Cyclodipeptide synthases (CDPSs) are a family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) to synthetize cyclodipeptides, which are precursors of many secondary metabolites with diverse biological functions 1,2. The first member of this family was identified in 2002 during characterization of the albonoursin biosynthetic pathway in Streptomyces noursei and called AlbC 3. Three CDPSs are structurally characterized and all three share a common architecture, consisting of a monomer containing a Rossmann fold domain 4-7. The CDPSs display strong structural similarity to the catalytic domains of class Ic aminoacyl tRNA synthetases (aaRSs), suggesting that CDPSs evolved from the class Ic of aaRSs 1. However, there are several significant differences between CDPSs and class Ic aaRSs. The ATP-binding motifs present in aaRSs are not present in CDPSs, since there is no need to activate amino acids in CDPSs, and CDPSs are active as monomers in contrast to the TyrRS and TrpRS that function as homodimers. The catalytic mechanism has been extensively studied experimentally for the structurally characterized CDPSs, and a structure mimicking a reaction intermediate was obtained for AlbC 7. It was demonstrated that AlbC uses Phe-tRNA Phe and Leu-tRNA Leu (or a second Phe-tRNA Phe) as substrates for a ping-pong mechanism involving the formation of two successive acyl-enzyme intermediates 1. The catalytic reaction starts with the binding of the first aa-tRNA and the transfer of its aminoacyl moiety to a conserved serine residue leading to the formation of an aminoacyl enzyme intermediate. For the second step, the tRNA Phe part of the first substrate dissociates from AlbC and a second aa-tRNA binds to the enzyme. The phenylalanyl-AlbC reacts with the second aa-tRNA to form a dipeptidyl-AlbC intermediate. In the final step, the target cyclodipeptide is obtained through intramolecular cyclization. Residues important for the reaction in AlbC were identified through site-directed mutagenesis and chemical biology studies 5,7. These residues, apart from S37, the conserved residue that accepts the aminoacyl group, are Y202, Y178, E182, N40, and H203. The residues Y178 and E182 are involved in the stabilization of the aminoacyl moiety of the first substrate (named Phe1) throughout the catalytic cycle as suggested by the crystal structure of the diphenylalanyl-enzyme intermediate mimic 7. E182 was also suggested to act as a general catalytic base during the formation of the dipeptidyl-enzyme by deprotonating the ammonium group of the aminoacyl-enzyme

Cyclodipeptide Synthases of the NYH Subfamily Recognize tRNA Using an α-Helix Enriched with Positive Residues

International audienceCyclodipeptide synthases (CDPSs) perform nonribosomal protein synthesis using two aminoacyl-tRNA substrates to produce cyclodipeptides. There is no available structural detail on the CDPS:tRNA interaction to date. Using AlbC, a CDPS that produces cyclo(L-Phe-L-Phe), the interaction between AlbC with 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 alpha helix, α4, significantly contribute to the binding free energy of AlbC to tRNA. Individual residue contributions to the tRNA binding free energy of the discovered binding mode explain well 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 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 tRNA interacting proteins