RESUMEN
The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.
Asunto(s)
Antibacterianos , Bacterias Gramnegativas , Antibacterianos/química , Bacterias Gramnegativas/metabolismo , Kanamicina Quinasa/química , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , PéptidosRESUMEN
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.
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Proteína Transportadora de Acilo , Sintasas Poliquetidas , Sintasas Poliquetidas/metabolismo , Microscopía por Crioelectrón , Sitios de Unión , Dominio Catalítico , Proteína Transportadora de Acilo/metabolismoRESUMEN
The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor.
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Metaloendopeptidasas , Factores de Virulencia , Humanos , Factores de Virulencia/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Pseudomonas aeruginosa/metabolismo , Escherichia coli/metabolismo , VirulenciaRESUMEN
Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexesâThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His ß-hydroxylationâa modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.
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Deshidratación , Señales de Clasificación de Proteína , Histidina , Humanos , Péptidos/química , Procesamiento Proteico-PostraduccionalRESUMEN
D-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of L-amino acids into their D-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a D-aspartate (D-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/ß-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of L-Asp to D-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH-substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.
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Racemasas y Epimerasas/química , Racemasas y Epimerasas/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Evolución Molecular , Modelos Moleculares , Familia de Multigenes , Péptidos Cíclicos/metabolismo , Conformación Proteica , Pliegue de Proteína , Racemasas y Epimerasas/genética , Streptomyces/enzimología , Streptomyces/genética , Especificidad por SustratoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Covering: 1950s up to the end of 2020Bottromycins are a class of macrocyclic peptide natural products that are produced by several Streptomyces species and possess promising antibacterial activity against clinically relevant multidrug-resistant pathogens. They belong to the ribosomally synthesised and post-translationally modified peptide (RiPP) superfamily of natural products. The structure contains a unique four-amino acid macrocycle formed via a rare amidine linkage, C-methylation and a D-amino acid. This review covers all aspects of bottromycin research with a focus on recent years (2009-2020), in which major advances in total synthesis and understanding of bottromycin biosynthesis were achieved.
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Antibacterianos/farmacología , Productos Biológicos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Procesamiento Proteico-PostraduccionalRESUMEN
Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
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Biología Computacional/métodos , Enzimas/metabolismo , Péptidos/química , Péptidos/metabolismo , Ingeniería de Proteínas/métodos , Productos Biológicos/química , Productos Biológicos/clasificación , Productos Biológicos/metabolismo , Enzimas/química , Hidroxilación , Metilación , Péptidos/clasificación , Péptidos/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/fisiología , Ribosomas/metabolismoRESUMEN
Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.
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Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Familia de Multigenes , Oxidación-Reducción , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Procesamiento Proteico-Postraduccional , Estereoisomerismo , Tiazoles/químicaRESUMEN
The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis.
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Amidohidrolasas/química , Proteínas Bacterianas/química , Amidohidrolasas/genética , Proteínas Bacterianas/genética , Biocatálisis , Mutación , Péptidos Cíclicos/química , Streptomyces/enzimologíaRESUMEN
Covering: up to 02/2019 This review covers the role of protein-protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes. The genomic organization of RiPP systems usually allows the expression of each biosynthetic enzyme as an individual unit, which is in stark contrast to the giant assembly lines found in non-ribosomal peptide and polyketide synthesis systems. Evidence is mounting however that the formation of multi-enzyme complexes is critical for efficient RiPPs biosynthesis and that these complexes may be involved in substrate channeling or conformational sampling. In some pathways, polyfunctional enzymes have evolved, which can be viewed as perpetual protein complexes. We summarize what is currently known on enzyme complexes in RiPP systems for lasso peptides, cyanobactins, linear azolic peptides, thiopeptides, and lanthipeptides.
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Complejos Multiproteicos/metabolismo , Péptidos/metabolismo , Ribosomas/metabolismo , Complejos Multiproteicos/química , Biosíntesis de Péptidos , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for α-galactoside-terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa We also investigated the utility of PllA as a probe for detecting α-galactosides. The α-Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA's high specificity for α-galactoside-containing ligands, and we show that PllA can be used to visualize the α-Gal epitope on porcine tissues.
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Galactósidos/metabolismo , Glicoconjugados/metabolismo , Lectinas/metabolismo , Photorhabdus/metabolismo , Secuencia de Aminoácidos , Animales , Pruebas de Hemaglutinación , Lectinas/química , Lectinas/aislamiento & purificación , Sondas Moleculares , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
To combat the rise of antimicrobial resistance, the discovery of new antibiotics is paramount. Albicidin and cystobactamid are related natural product antibiotics with potent activity against Gram-positive and, crucially, Gram-negative pathogens. AlbA has been reported to neutralize albicidin by binding it with nanomolar affinity. To understand this potential resistance mechanism, we determined structures of AlbA and its complex with albicidin. The structures revealed AlbA to be comprised of two domains, each unexpectedly resembling the multiantibiotic neutralizing protein TipA. Binding of the long albicidin molecule was shared pseudosymmetrically between the two domains. The structure also revealed an unexpected chemical modification of albicidin, which we demonstrate to be promoted by AlbA, and to reduce albicidin potency; we propose a mechanism for this reaction. Overall, our findings suggest that AlbA arose through internal duplication in an ancient TipA-like gene, leading to a new binding scaffold adapted to the sequestration of long-chain antibiotics.
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Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Asparagina/análogos & derivados , Asparagina/química , Asparagina/farmacología , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Ciclización , Farmacorresistencia Bacteriana Múltiple/fisiología , Evolución Molecular , Klebsiella oxytoca/química , Pruebas de Sensibilidad Microbiana , Mutación , Nitrocompuestos/química , Nitrocompuestos/farmacología , Compuestos Orgánicos/química , Compuestos Orgánicos/metabolismo , Compuestos Orgánicos/farmacología , Unión Proteica , Xanthomonas/química , Xanthomonas/metabolismoRESUMEN
The YcaO superfamily of proteins catalyzes the phosphorylation of peptide backbone amide bonds, which leads to the formation of azolines and azoles in ribosomally synthesized and post-translationally modified peptides (RiPPs). Bottromycins are RiPPs with potent antimicrobial activity, and their biosynthetic pathway contains two divergent, stand-alone YcaO enzymes, IpoC and PurCD. From an untargeted metabolomics approach, it had been suggested that PurCD acts with a partner protein to form the 12-membered macroamidine unique to bottromycins. Here we report the biochemical characterization of IpoC and PurCD. We demonstrate that IpoC installs a cysteine-derived thiazoline, whereas PurCD alone is sufficient to create the macroamidine structure. Both enzymes are catalytically promiscuous, and we generated 10 different macroamidines. Our data provide important insights into the versatility of YcaO enzymes, their ability to utilize different nucleophiles and provide a framework for the creation of novel bottromycin derivatives with enhanced bioactivity.
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Amidinas/química , Compuestos Macrocíclicos/química , Secuencia de Aminoácidos , Catálisis , Ciclización , Estructura Molecular , Biosíntesis de Péptidos , Péptidos/química , Péptidos/genética , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/químicaRESUMEN
Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the ß-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.
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Proteínas Bacterianas/química , Péptidos Cíclicos/química , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Cianobacterias/química , Cianobacterias/metabolismo , Ciclización , Cisteína/química , Cisteína/metabolismo , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Tiazoles/química , Tiazoles/metabolismoRESUMEN
The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N-terminal "leader" sequence. We report herein the structural and biochemical characterization of BotP, a leucyl-aminopeptidase-like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro- mycin.
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Leucil Aminopeptidasa/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Especificidad por SustratoRESUMEN
Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids.
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Azoles/metabolismo , Oxidorreductasas/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos Cíclicos/biosíntesis , Liasas de Fósforo-Oxígeno/metabolismo , Azoles/química , Conformación Molecular , Oxidorreductasas/química , Péptido Hidrolasas/química , Péptidos Cíclicos/química , Liasas de Fósforo-Oxígeno/químicaRESUMEN
Hydrophilic polymers have found ubiquitous use in drug delivery and novel polymer materials to advance drug delivery systems are highly sought after. Herein, an amylose mimic (PEGose) was combined with poly(lactic acid) (PLA) in an amphiphilic block copolymer to form PEG-free nanoparticles as an alternative to PEG-based nanomedicines. The block copolymer self-assembled into 150-200 nm particles with a narrow dispersity in aqueous environment. The formed nanoparticles were capable of encapsulation, the sustained release of both hydrophilic and hydrophobic dyes. Moreover, the nanoparticles were found to be remarkably stable and had a very low cytotoxicity and a high propensity to penetrate cells. These results highlight the potential of PEGose-b-PLA to be used in drug delivery with a new hydrophilic building block.