RESUMEN
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways is challenging. Here, it is reported that peptide/protein L-aspartyl O-methyltransferases (PAMTs) present in several RiPP subclasses are highly homologous. Importantly, it is discovered that the apparent evolutionary transmission of the PAMT gene to unrelated RiPP subclasses can serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that homologous PAMTs convert aspartate to isoaspartate via aspartyl-O-methyl ester and aspartimide intermediates, and often require cyclic or hairpin-like structures for modification. By conducting homology-based bioinformatic analysis of PAMTs, over 2,800 biosynthetic gene clusters (BGCs) are identified for known RiPP subclasses in which PAMTs install a secondary modification, and over 1,500 BGCs where PAMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pamtides. The results suggest that the genome mining of proteins with secondary biosynthetic roles can be an effective strategy for discovering novel biosynthetic pathways of RiPPs through the principle of "guilt by association".
Asunto(s)
Ácido Isoaspártico , Metiltransferasas , Ácido Isoaspártico/genética , Ácido Isoaspártico/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Procesamiento Proteico-Postraduccional , Péptidos/metabolismoRESUMEN
Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on RiPPs, collectively named "cyptides". Homology-based genome mining for P450 macrocyclases revealed 19 novel groups of homologous biosynthetic gene clusters (BGCs) with distinct aromatic residue patterns in the precursor peptides. Using the P450-modified precursor peptides heterologously coexpressed with corresponding P450s in Escherichia coli, we determined the NMR structures of three novel biaryl-containing peptidesâthe enzymatic products, roseovertin (1), rubrin (2), and lapparbin (3)âand confirmed the formation of three unprecedented or rare biaryl linkages: Trp C-7'-to-His N-τ in 1, Trp C-7'-to-Tyr C-6 in 2, and Tyr C-6-to-Trp N-1' in 3. Biochemical characterization indicated that certain P450s in these pathways have a relaxed substrate specificity. Overall, our studies suggest that P450 macrocyclases have evolved to create diverse biaryl linkages in RiPPs, promoting the exploration of a broader chemical space for biaryl-containing peptides encoded in bacterial genomes.
RESUMEN
Cihunamidesâ A-D (1-4), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1-4 were elucidated by 1 H, 13 C, and 15 N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C-N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochromeâ P450 enzyme and a precursor peptide. Heterologous co-expression of the core genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp-Trp linkage. Cihunamides do not display the non-canonical atropisomerism shown in tryptorubins, which are the founding members of the "atropitide" family. Therefore, we propose to use a new RiPP family name, "bitryptides", for cihunamides, tryptorubins, and their congeners, wherein the Trp-Trp linkages define the structural class rather than non-canonical atropisomerism.
Asunto(s)
Productos Biológicos , Péptidos , Péptidos/química , Biología Computacional , Procesamiento Proteico-Postraduccional , Genoma , Sistema Enzimático del Citocromo P-450/genéticaRESUMEN
Backbone N-methylation is one of the prominent peptide modifications that can greatly enhance the pharmacological properties of a peptide. Naturally occurring backbone N-methylated peptides are produced via nonribosomal or ribosomal pathways, the latter of which was only recently identified in the borosin family of ribosomally synthesized and post-translationally modified peptides. Although previous bioinformatic analyses have revealed new putative genes for borosin biosynthesis, the natural scope of structural and biosynthetic diversity of the borosin family has not been thoroughly explored. Here, we report a comprehensive overview of the borosin family of peptide natural products. Using a genome mining approach, we identified more than 1400 new putative biosynthetic gene clusters for borosins and demonstrated that, unlike those previously reported, most of them are found in bacterial genomes and encode a precursor peptide unfused to its cognate methyltransferase enzyme. Biochemical analysis confirmed the backbone N-methylation of the precursor peptide in trans in eight enzyme-precursor pairs and revealed two novel types of enzyme-recognizing sequences in the precursor peptide. This work significantly expands the biosynthetic diversity of borosins and paves the way for the enzymatic production of diverse backbone N-methylated peptides.
Asunto(s)
Bacterias/metabolismo , Metiltransferasas/metabolismo , Péptidos/metabolismo , Bacterias/genética , Biología Computacional/métodos , Genoma Bacteriano , Espectrometría de Masas/métodos , Metilación , Familia de Multigenes , Péptidos/genética , Procesamiento Proteico-Postraduccional , Ribosomas/metabolismo , Especificidad por SustratoRESUMEN
Methods that can randomly introduce mutations in the microbial genome have been used for classical genetic screening and, more recently, the evolutionary engineering of microbial cells. However, most methods rely on either cell-damaging agents or disruptive mutations of genes that are involved in accurate DNA replication, of which the latter requires prior knowledge of gene functions, and thus, is not easily transferable to other species. In this study, we developed a new mutator for in vivo mutagenesis that can directly modify the genomic DNA. Mutator protein, MutaEco, in which a DNA-modifying enzyme is fused to the α-subunit of Escherichia coli RNA polymerase, increases the mutation rate without compromising the cell viability and accelerates the adaptive evolution of E. coli for stress tolerance and utilization of unconventional carbon sources. This fusion strategy is expected to accommodate diverse DNA-modifying enzymes and may be easily adapted to various bacterial species.
Asunto(s)
Escherichia coli , Técnicas Genéticas , Replicación del ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , MutagénesisRESUMEN
ω-Ester-containing peptides (OEPs) are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing intramolecular ω-ester or ω-amide bonds. Although their distinct side-to-side connections may create considerable topological diversity of multicyclic peptides, it is largely unknown how diverse ring patterns have been developed in nature. Here, using genome mining of biosynthetic enzymes of OEPs, we identified genes encoding nine new groups of putative OEPs with novel core consensus sequences, disclosing a total of â¼1500 candidate OEPs in 12 groups. Connectivity analysis revealed that OEPs from three different groups contain novel tricyclic structures, one of which has a distinct biosynthetic pathway where a single ATP-grasp enzyme produces both ω-ester and ω-amide linkages. Analysis of the enzyme cross-reactivity showed that, while enzymes are promiscuous to nonconserved regions of the core peptide, they have high specificity to the cognate core consensus sequence, suggesting that the enzyme-core pair has coevolved to create a unique ring topology within the same group and has sufficiently diversified across different groups. Collectively, our results demonstrate that the diverse ring topologies, in addition to diverse sequences, have been developed in nature with multiple ω-ester or ω-amide linkages in the OEP family of RiPPs.
Asunto(s)
Adenosina Trifosfato/química , Enzimas/química , Evolución Química , Genoma , Péptidos/química , Ésteres/químicaRESUMEN
The modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) enhances their engineering potential for exploring new structures and biological functions. The ω-ester-containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to improve the engineering potential of RiPPs, we present here the idea that the multidomain architecture of an OEP, plesiocin, can be exploited to create a bifunctional modified peptide. Characterization of plesiocin variants revealed that strong chymotrypsin inhibition relies on the bicyclic structure of the domain in which a leucine residue in the hairpin loop functions as a specificity determinant. Four domains of plesiocin promote simultaneous binding of multiple enzymes, where the C-terminal domain binds chymotrypsin most efficiently. Using this information, we successfully engineered a plesiocin variant in which two different domains inhibit chymotrypsin and trypsin. This result suggests that the multidomain architecture of OEPs is a useful platform for engineering multifunctional hybrid RiPPs.
Asunto(s)
Quimotripsina/antagonistas & inhibidores , Péptidos/química , Ingeniería de Proteínas , Vías Biosintéticas/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Quimotripsina/química , Clonación Molecular , Escherichia coli/genética , Ésteres/química , Péptidos/genética , Péptidos/aislamiento & purificación , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica/genética , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , Ribosomas/química , Ribosomas/genética , Tripsina/química , Tripsina/genética , Inhibidores de Tripsina/químicaRESUMEN
Microviridins are ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain multiple intramolecular ω-ester or ω-amide crosslinks between two side chains in peptides. This type of the side-to-side macrocyclization may generate diverse structures with distinct topology and ring sizes, but the majority of the microviridin-like RiPPs present only a single consensus sequence with a tricyclic architecture. Here, we expanded the natural diversity of the microviridin-like modified peptides by determining the crosslinking connectivity of a new modified peptide, mTgnA and its homologous RiPPs, which we named the thuringinin group. Members of the thuringinin group have core motifs with a distinct consensus sequence, which is transformed to a novel hairpin-like bicyclic structure by the cognate ATP-grasp enzyme. We suggest that the microviridin-like RiPPs naturally have novel sequences and architectures beyond those found in microviridins and comprise a larger RiPP family, termed omega-ester containing peptides (OEPs).
Asunto(s)
Péptidos Cíclicos/química , Secuencia de Aminoácidos , Ésteres/química , Procesamiento Proteico-PostraduccionalRESUMEN
Macrocyclization of peptides is often employed to generate novel structures and biological activities in the biosynthesis of natural products and drug discovery. The enzymatic cross-linking of two side chains in a peptide via an ester or amide has a high potential for making topologically diverse cyclic peptides but is found with only a single consensus sequence in the microviridin class of natural products. Here, we report that a peptide with a new sequence pattern can be enzymatically cross-linked to make a novel microviridin-like peptide, plesiocin, which contains four repeats of a distinct hairpin-like bicyclic structure and shows strong inhibition of proteases. A single ATP-grasp enzyme binds to a leader peptide, of which only 13 residues are required for binding, and performs eight esterification reactions on the core peptide. We also demonstrate that the combination of tandem mass spectrometry and an ester-specific reaction greatly facilitates the determination of connectivity. We suggest that the enzymatic cross-linking of peptide side chains can generate more diverse structures in nature or by engineering.