Flexizyme-aminoacylated shortened tRNAs demonstrate that only the aminoacylated acceptor arms of the two tRNA substrates are required for cyclodipeptide synthase activity.

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.

In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli.

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-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized.

Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches.

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.

Author Correction: Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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.

Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids.

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.

Structural basis for partition of the cyclodipeptide synthases into two subfamilies.

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.

A Comprehensive Overview of the Cyclodipeptide Synthase Family Enriched with the Characterization of 32 New Enzymes.

Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored.

Incorporation of Non-canonical Amino Acids into 2,5-Diketopiperazines by Cyclodipeptide Synthases.

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 in 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.

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.

Analysis of 51 cyclodipeptide synthases reveals the basis for substrate specificity.

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.

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

Cyclodipeptide 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.

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.

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.

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.

Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.

Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs).

The nonribosomal synthesis of diketopiperazines in tRNA-dependent cyclodipeptide synthase pathways.

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.

Nonribosomal peptide synthesis in animals: the cyclodipeptide synthase of Nematostella.

Cyclodipeptide synthases (CDPSs) are small enzymes structurally related to class-I aminoacyl-tRNA synthetases (aaRSs). They divert aminoacylated tRNAs from their canonical role in ribosomal protein synthesis, for cyclodipeptide formation. All the CDPSs experimentally characterized to date are bacterial.

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.

Dynamic combinatorial self-assembly of cyclophilin hCyp-18 ligands through oxorhenium coordination.

The dynamic combinatorial assembly of independent modules A and B through oxorhenium(V) coordination by a NS2+S motif in the presence of cyclophilin hCyp-18-an important peptidyl-prolyl isomerase-was investigated. Increasing glutathione (GSH) concentrations were used to dissociate [ARe(V)OB] complexes that displayed low affinity for hCyp-18. Conversely, coordinates that displayed submicromolar affinities for hCyp-18 were protected against thiol exchange and could be detected by LC-MS.

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17.

Biochemical properties and cellular localization of Plasmodium falciparum protein disulfide isomerase.

We have previously reported the isolation of a 52,000 M(r) protein (Pf52) displaying consensus sequences for thiol:disulfide oxidoreductases. Pf52 therefore represents the plasmodial protein disulfide isomerase (PDI). It has been renamed PfPDI and correlates to MAL8P1.

A tandem of SH3-like domains participates in RNA binding in KIN17, a human protein activated in response to genotoxics.

The human KIN17 protein is an essential nuclear protein conserved from yeast to human and expressed ubiquitously in mammals. Suppression of Rts2, the yeast equivalent of gene KIN17, renders the cells unviable, and silencing the human KIN17 gene slows cell growth dramatically. Moreover, the human gene KIN17 is up-regulated following exposure to ionizing radiations and UV light, depending on the integrity of the human global genome repair machinery.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.

Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae.

We studied the interaction of several metal ions with the copper chaperone from Enterococcus hirae (EhCopZ). We show that the stoichiometry of the protein-metal complex varies with the experimental conditions used. At high concentration of the protein in a noncoordinating buffer, a dimer, (EhCopZ)2-metal, was formed.

Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry.

Metallochaperones are soluble proteins involved in metal transport and regulation in vivo. Copper metallochaperones belong to a structural family of metal binding domains displaying a ferredoxin-like fold (betaalphabetabetaalphabeta) and a consensus metal-binding motif MXCXXC. The metal-binding selectivities for this class of proteins are poorly documented so far.