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1.
Proc Natl Acad Sci U S A ; 117(1): 371-380, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871149

RESUMO

Microbial natural products represent a rich resource of evolved chemistry that forms the basis for the majority of pharmacotherapeutics. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a particularly interesting class of natural products noted for their unique mode of biosynthesis and biological activities. Analyses of sequenced microbial genomes have revealed an enormous number of biosynthetic loci encoding RiPPs but whose products remain cryptic. In parallel, analyses of bacterial metabolomes typically assign chemical structures to only a minority of detected metabolites. Aligning these 2 disparate sources of data could provide a comprehensive strategy for natural product discovery. Here we present DeepRiPP, an integrated genomic and metabolomic platform that employs machine learning to automate the selective discovery and isolation of novel RiPPs. DeepRiPP includes 3 modules. The first, NLPPrecursor, identifies RiPPs independent of genomic context and neighboring biosynthetic genes. The second module, BARLEY, prioritizes loci that encode novel compounds, while the third, CLAMS, automates the isolation of their corresponding products from complex bacterial extracts. DeepRiPP pinpoints target metabolites using large-scale comparative metabolomics analysis across a database of 10,498 extracts generated from 463 strains. We apply the DeepRiPP platform to expand the landscape of novel RiPPs encoded within sequenced genomes and to discover 3 novel RiPPs, whose structures are exactly as predicted by our platform. By building on advances in machine learning technologies, DeepRiPP integrates genomic and metabolomic data to guide the isolation of novel RiPPs in an automated manner.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Descoberta de Drogas/métodos , Peptídeos/isolamento & purificação , Software , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Produtos Biológicos/metabolismo , Genômica/métodos , Aprendizado de Máquina , Metabolômica/métodos , Biossíntese Peptídica/genética , Peptídeos/genética , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
2.
Proc Natl Acad Sci U S A ; 116(48): 24049-24055, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31719203

RESUMO

Enzymes that generate ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products have garnered significant interest, given their ability to produce large libraries of chemically diverse scaffolds. Such RiPP biosynthetic enzymes are predicted to bind their corresponding peptide substrates through sequence-specific recognition of the leader sequence, which is removed after the installation of posttranslational modifications on the core sequence. The conservation of the leader sequence within a given RiPP class, in otherwise disparate precursor peptides, further supports the notion that strict sequence specificity is necessary for leader peptide engagement. Here, we demonstrate that leader binding by a biosynthetic enzyme in the lasso peptide class of RiPPs is directed by a minimal number of hydrophobic interactions. Biochemical and structural data illustrate how a single leader-binding domain can engage sequence-divergent leader peptides using a conserved motif that facilitates hydrophobic packing. The presence of this simple motif in noncognate peptides results in low micromolar affinity binding by binding domains from several different lasso biosynthetic systems. We also demonstrate that these observations likely extend to other RiPP biosynthetic classes. The portability of the binding motif opens avenues for the engineering of semisynthetic hybrid RiPP products.


Assuntos
Modelos Moleculares , Biossíntese Peptídica , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Processamento de Proteína Pós-Traducional
3.
Nat Commun ; 10(1): 4563, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594941

RESUMO

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.


Assuntos
Antibacterianos/farmacologia , Azóis/farmacologia , Agentes de Controle Biológico/farmacologia , Peptídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Azóis/química , Azóis/metabolismo , Agentes de Controle Biológico/química , Agentes de Controle Biológico/metabolismo , Microscopia Crioeletrônica , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Testes de Sensibilidade Microbiana , Família Multigênica , Biossíntese Peptídica/genética , Peptídeos/química , Peptídeos/metabolismo , Phaseolus/microbiologia , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/ultraestrutura , Rhizobium/genética , Rhizobium/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Especificidade da Espécie , Simbiose
4.
ACS Chem Biol ; 14(9): 1981-1989, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31449382

RESUMO

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the ß- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the ß-carbon of Asp (S-Cß). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Biossíntese Peptídica/fisiologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Paenibacillus polymyxa/enzimologia , Peptídeos/química , Especificidade por Substrato
5.
Science ; 365(6450): 280-284, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31320540

RESUMO

Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA-dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid-derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid-derived natural products.


Assuntos
Aminoácidos/química , Produtos Biológicos/metabolismo , Biossíntese Peptídica , Aminoácidos/metabolismo , Produtos Biológicos/química , Escherichia coli , Família Multigênica , Peptídeos/química , Peptídeos/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Pirróis/química , Quinolinas/química
6.
J Biol Chem ; 294(40): 14512-14525, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31337708

RESUMO

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.


Assuntos
Bacteriocinas/química , Clostridiales/genética , Clostridium perfringens/genética , Microbioma Gastrointestinal/genética , Peptídeos/genética , Sequência de Aminoácidos/genética , Bacteriocinas/biossíntese , Bacteriocinas/genética , Clostridiales/enzimologia , Clostridium perfringens/química , Clostridium perfringens/patogenicidade , Humanos , Família Multigênica/genética , Biossíntese Peptídica/genética , Peptídeos/química , Processamento de Proteína Pós-Traducional/genética , Ribossomos/genética , Motivo Estéril alfa/genética , Sulfetos/química , Simbiose/genética
7.
Molecules ; 24(14)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336853

RESUMO

Angiotensin I-converting enzyme (ACE) is a paramount therapeutic target to treat hypertension. ACE inhibitory peptides derived from food protein sources are regarded as safer alternatives to synthetic antihypertensive drugs for treating hypertension. Recently, marine organisms have started being pursued as sources of potential ACE inhibitory peptides. Marine organisms such as fish, shellfish, seaweed, microalgae, molluscs, crustaceans, and cephalopods are rich sources of bioactive compounds because of their high-value metabolites with specific activities and promising health benefits. This review aims to summarize the studies on peptides from different marine organisms and focus on the potential ability of these peptides to inhibit ACE activity.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Organismos Aquáticos/metabolismo , Biossíntese Peptídica , Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Biomarcadores , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Peptídeos/química
8.
ACS Chem Biol ; 14(7): 1619-1627, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31188556

RESUMO

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3D-interlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida fusca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal ß-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal ß-sheet of TfuB1, as an additional ß strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the ß-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the ß-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.


Assuntos
Actinobacteria/química , Proteínas de Bactérias/química , Peptídeos/química , Sinais Direcionadores de Proteínas , Cristalografia por Raios X , Modelos Moleculares , Biossíntese Peptídica , Conformação Proteica , Processamento de Proteína Pós-Traducional
9.
PLoS One ; 14(5): e0217369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125361

RESUMO

Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the ß-cyclodextrin (ß-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized ß-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição/métodos , Ciclodextrinas/química , Cisteína Endopeptidases/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Dendrímeros/síntese química , Dendrímeros/química , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Biossíntese Peptídica , Peptídeos/síntese química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(22): 10658-10663, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31088971

RESUMO

Ribozymes synthesize proteins in a highly regulated local environment to minimize side reactions caused by various competing species. In contrast, it is challenging to prepare synthetic polypeptides from the polymerization of N-carboxyanhydrides (NCAs) in the presence of water and impurities, which induce monomer degradations and chain terminations, respectively. Inspired by natural protein synthesis, we herein report the preparation of well-defined polypeptides in the presence of competing species, by using a water/dichloromethane biphasic system with macroinitiators anchored at the interface. The impurities are extracted into the aqueous phase in situ, and the localized macroinitiators allow for NCA polymerization at a rate which outpaces water-induced side reactions. Our polymerization strategy streamlines the process from amino acids toward high molecular weight polypeptides with low dispersity by circumventing the tedious NCA purification and the demands for air-free conditions, enabling low-cost, large-scale production of polypeptides that has potential to change the paradigm of polypeptide-based biomaterials.


Assuntos
Aminoácidos/química , Anidridos/química , Peptídeos , Polimerização , Cinética , Cloreto de Metileno/química , Modelos Biológicos , Peso Molecular , Biossíntese Peptídica , Peptídeos/síntese química , Peptídeos/química , Água/química
11.
Australas J Dermatol ; 60(4): e267-e271, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30941744

RESUMO

Peptides stimulating synthesis of extracellular matrix are now commonly used in the production of anti-ageing cosmetics. However, much uncertainty still exists about the methodology of their clinical assessment. The aim of the study was to review the literature for clinical study designs assessing the efficacy of these peptides. The authors searched systematically publications indexed in PubMed, Scope and Web of Science, according to the PRISMA protocol. Altogether 12 scientific papers, reporting results of 15 independent studies were identified. Out of these 15 studies, only six used a placebo control. Double-blinding was applied in five out of 15 studies. Nine studies were based on female-only populations. For the product performance evaluation, most of the studies (10 out of the 15) used image-based methods. The literature on the topic is sparse. The studies carried out so far have many methodological limitations. Most of the clinical experiments hitherto conducted were non-double-blind and used no placebo control. There is a need for better planned and controlled clinical trials in this area.


Assuntos
Cosméticos , Matriz Extracelular/metabolismo , Biossíntese Peptídica , Envelhecimento da Pele/fisiologia , Administração Tópica , Proteínas da Matriz Extracelular/metabolismo , Humanos , Peptídeos/metabolismo
12.
Nucleic Acids Res ; 47(9): 4624-4637, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30916321

RESUMO

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.


Assuntos
Produtos Biológicos/metabolismo , Biossíntese Peptídica/genética , Peptídeos Cíclicos/genética , Peptídeos/genética , Actinobacteria/química , Actinobacteria/genética , DNA Polimerase Dirigida por DNA/genética , Genoma/genética , Peptídeos/química , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional/genética , Ribossomos/genética , Streptomyces/genética , Streptomyces/metabolismo , Tioamidas
13.
Appl Microbiol Biotechnol ; 103(6): 2649-2664, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30707253

RESUMO

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Carboxil e Carbamoil Transferases/metabolismo , Metiltransferases/metabolismo , Peptídeos/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/isolamento & purificação , Produtos Biológicos , Vias Biossintéticas , Carboxil e Carbamoil Transferases/isolamento & purificação , Metilação , Metiltransferases/isolamento & purificação , Biossíntese Peptídica , Peptídeos Cíclicos , Fosforilação , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
14.
Amino Acids ; 51(4): 669-678, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30758725

RESUMO

The synthesis of α/ß-chimeras comprises peptide bond formation from α- to ß-, from ß- to ß-, and from ß- to α-amino acid residues. The fine-tuned solid phase synthesis of -GXXG- chimera peptides containing the simplest achiral α-amino acid glycine and two cyclic SAAs of different ring size [X denoting cyclic ß-Sugar Amino Acids (ß-SAA)] is reported, variants containing Fmoc-RibAFU(ip)-OH a furanoid-, and Fmoc-GlcAPU(Me)-OH a pyranoid-type structural "Lego-element". Systematic search for the best coupling strategy with both H-ß-SAA-OHs is described, including the comparison of the different coupling reagents and conditions. Selecting the optimal reagent (from commonly used PyBOP, HATU and HOBt) was assisted by time-resolved 1H-NMR: formation and stability of the Fmoc protected active esters were compared. We found that PyBOP is the best choice for successfully coupling both H-ß-SAA-OH prototypes. The present comparative results open a reasonable route for building efficiently various -ß-SAA- containing homo- and heterooligomers.


Assuntos
Aminoácidos Cíclicos/química , Amino Açúcares/química , Biossíntese Peptídica , Fragmentos de Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida
15.
Amino Acids ; 51(4): 661-667, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30798465

RESUMO

Selenocysteine (Sec) residue cannot be directly attached to a peptide sequence unless the selenol form is protected beforehand and several problems have been reported in the preparation of Sec building blocks. In this article a series of selenocystine, the oxidized form of Sec, containing peptides has been synthesized using a new methodology, where Boc-NH-chloroalanine is coupled with methyl ester protected residues (Ala, Met, Phe) using DCC/HOBt as the coupling reagents providing di- and tripeptides. Further, the treatment of disodium diselenide with chloroalanine peptides (Boc-ClAla-Ala-OMe, Boc-ClAla-Met-OMe and Boc-ClAla-Ala-Phe-OMe) afforded the respective selenocystine-containing peptides (Boc-Sec-Ala-OMe, Boc-Sec-Met-OMe and Boc-Sec-Ala-Phe-OMe).


Assuntos
Aminoácidos/química , Cistina/análogos & derivados , Compostos Organosselênicos/química , Biossíntese Peptídica , Fragmentos de Peptídeos/química , Selenoproteínas/síntese química , Cistina/química , Modelos Químicos , Estrutura Molecular , Fragmentos de Peptídeos/síntese química
16.
Chembiochem ; 20(11): 1357-1364, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30618145

RESUMO

Expanding the genetic code of organisms by incorporating noncanonical amino acids (ncAAs) into target proteins through the suppression of stop codons in vivo has profoundly impacted how we perform protein modification or detect proteins and their interaction partners in their native environment. Yet, with genetic code expansion strategies maturing over the past 15 years, new applications that make use-or indeed repurpose-these techniques are beginning to emerge. This Concept article highlights three of these developments: 1) The incorporation of ncAAs for the biosynthesis and selection of bioactive macrocyclic peptides with novel ring architectures, 2) synthetic biocontainment strategies based on the addiction of microorganisms to ncAAs, and 3) enzyme design strategies, in which ncAAs with unique functionalities enable the catalysis of new-to-nature reactions. Key advances in all three areas are presented and potential future applications discussed.


Assuntos
Aminoácidos/química , Escherichia coli , Microrganismos Geneticamente Modificados , Peptídeos Cíclicos , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Biossíntese Peptídica , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Biologia Sintética/métodos
17.
ACS Chem Biol ; 14(2): 204-213, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30648860

RESUMO

Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.


Assuntos
Aminoácidos/metabolismo , Biossíntese de Proteínas , Aminoácidos/química , Guanosina Trifosfato/metabolismo , Hidrólise , Cinética , Biossíntese Peptídica , Fator Tu de Elongação de Peptídeos/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Reprodutibilidade dos Testes , Estereoisomerismo
18.
Appl Microbiol Biotechnol ; 103(6): 2731-2743, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30666364

RESUMO

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.


Assuntos
Proteínas de Bactérias/genética , Caseínas/metabolismo , Endopeptidases/genética , Lactobacillus delbrueckii/enzimologia , Peptídeos/metabolismo , Hidrolisados de Proteína/metabolismo , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Linhagem Celular , Endopeptidases/metabolismo , Humanos , Fatores Imunológicos/isolamento & purificação , Lactobacillus delbrueckii/genética , Lactococcus lactis/genética , Biossíntese Peptídica , Peptidil Dipeptidase A/metabolismo , Sinais Direcionadores de Proteínas , Proteólise
19.
Org Biomol Chem ; 17(5): 1027-1036, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30608100

RESUMO

The Basidiomycota, also called club fungi, comprise a diverse group of fungi. Basidiomycota are strongly related to ecosystem functioning along with human life. These fungi display a wide range of bioactivities, and some are known to produce of deadly toxins or hallucinogens. Some Basidiomycota have be used as medicinal mushrooms for thousands of years. Recently, the biosyntheses of several classes of natural products from Basidiomycota have been reported. Here, we review recent studies on the biosynthetic pathways and enzymes of bioactive natural products from Basidiomycota fungi, with a focus on terpenoids, alkaloids, ribosomally synthesized and post-translationally modified peptides (RiPPs), and polyketides.


Assuntos
Basidiomycota/metabolismo , Produtos Biológicos/metabolismo , Alcaloides/biossíntese , Vias Biossintéticas , Proteínas Fúngicas/metabolismo , Biossíntese Peptídica , Peptídeos/metabolismo , Policetídeos/metabolismo , Processamento de Proteína Pós-Traducional , Terpenos/metabolismo
20.
J Antibiot (Tokyo) ; 72(1): 22-33, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30315257

RESUMO

Novel benzoxaborole derivatives of azithromycin in which benzoxaborole residue is attached to the 4″-hydroxy-group of azithromycin have been synthesized. Antibacterial activity of synthesized derivatives in comparison with azithromycin was tested on a panel of Gram-positive and Gram-negative bacterial strains. All the investigated compounds demonstrated broad spectrum of antibacterial activity being in general more active against Gram-positive strains. New benzoxaborole derivatives of azithromycin demonstrated high activity against Streptococcus pyogenes ATCC 19615 and Propionibacterium acnes ATCC 6919 strains. Some of the new compounds were more active than azithromycin against Streptococcus pneumoniae ATCC 49619 strain or Enterococcus faecium strains. Using a reporter construct created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon pRFPCER-TrpL2A it has been demonstrated that the mechanism of action of azithromycin analogs is blocking nascent peptide in ribosome tunnel.


Assuntos
Antibacterianos/farmacologia , Azitromicina/análogos & derivados , Azitromicina/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Azitromicina/síntese química , Azitromicina/química , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Biossíntese Peptídica/efeitos dos fármacos
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