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1.
Chem Commun (Camb) ; 55(58): 8390-8393, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257394

RESUMO

Diverse bioactive alkaloids with a tryptophan 2,5-diketopiperazine (DKP) core and an annulated structure forming a methylated pyrroloindoline-DKP assembly have been isolated from various microbial sources. However, little is known about their biosynthesis. In this study, a novel indole C3 methyltransferase from Streptomyces sp. HPH0547 was discovered and characterized. Structural elucidation of the products revealed that this enzyme catalyzed unique pyrroloindoline cyclization in tryptophan-containing cyclodipeptides. This is the first C3 methyltransferase reported to catalyze pyrroloindoline cyclization in cyclic dipeptides, which provides a feasible and simple method to access diverse alkaloids.


Assuntos
Alcaloides/biossíntese , Proteínas de Bactérias/metabolismo , Dipeptídeos/biossíntese , Metiltransferases/metabolismo , Peptídeos Cíclicos/biossíntese , Streptomyces/enzimologia , Ciclização , Dicetopiperazinas/metabolismo , Modelos Químicos , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
2.
Appl Microbiol Biotechnol ; 103(11): 4467-4481, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989253

RESUMO

Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.


Assuntos
Antibacterianos/biossíntese , Bacillus/metabolismo , Proteínas de Bactérias/biossíntese , Vias Biossintéticas/genética , Lipopeptídeos/biossíntese , Engenharia Metabólica/métodos , Peptídeos Cíclicos/biossíntese , Proteínas Recombinantes/biossíntese , Bacillus/genética , Proteínas de Bactérias/genética , Expressão Gênica , Lipopeptídeos/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Família Multigênica , Peptídeos Cíclicos/genética , Proteínas Recombinantes/genética
3.
Microb Cell Fact ; 18(1): 68, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971238

RESUMO

BACKGROUND: Iturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon. RESULTS: In this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l-1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14 + 2H]2+, [C15 + 2H]2+, [C16 + 2H]2+ and [C17 + 2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l-1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l-1 by overexpression of a pleiotropic regulator DegQ. CONCLUSIONS: To our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14-C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.


Assuntos
Bacillus amyloliquefaciens/metabolismo , Engenharia Metabólica , Peptídeos Cíclicos/biossíntese , Antifúngicos , Bacillus amyloliquefaciens/genética , Vias Biossintéticas , Fermentação , Genoma Bacteriano , Lipopeptídeos/biossíntese , Óperon , Regiões Promotoras Genéticas , Transcrição Genética
4.
Appl Microbiol Biotechnol ; 103(10): 3931-3940, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30915503

RESUMO

Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.


Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas/tendências , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Tecnologia Farmacêutica/métodos , Antituberculosos/isolamento & purificação , Antituberculosos/metabolismo , Biotecnologia/métodos , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/isolamento & purificação , Tuberculose/tratamento farmacológico
5.
J Oleo Sci ; 67(10): 1307-1313, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30305561

RESUMO

Bacillus licheniformis NBRC 104464 produces a cyclic lipopeptide different from surfactin. After we performed liquid chromatography fractionation and purification, we used structural analyses to identify the cyclic lipopeptide as lichenysin. Surface tension measurements of lichenysin sodium salt in water yielded a critical micelle concentration (CMC) of 1.0×10-5 M. The surface tension at the CMC was 28.9 mN/m. Comparative analysis of Ca2+-influenced micellar aggregation of lichenysin and surfactin revealed that the formation rate of the lichenysin-Ca2+ complex aggregate remained low up to a [Ca2+]/[lichenysin] molar ratio of 80, whereas the surfactin-Ca2+ complex formed micellar aggregates at the same molar ratio. Further excessive addition of Ca2+ to the micellar solution of lichenysin induced higher turbidity than surfactin.


Assuntos
Bacillus licheniformis/metabolismo , Cálcio , Lipoproteínas/biossíntese , Lipoproteínas/química , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Cálcio/química , Cromatografia Líquida , Lipopeptídeos/química , Lipoproteínas/isolamento & purificação , Micelas , Peptídeos Cíclicos/isolamento & purificação , Soluções , Propriedades de Superfície , Tensão Superficial , Tensoativos/química
6.
Int J Food Microbiol ; 284: 11-21, 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29990635

RESUMO

In Aspergillus flavus, laeA affects cell morphology and contributes to the production of secondary metabolites (SMs) production including aflatoxin, cyclopiazonic acid, and aflatrem. Here, we investigated the function of this transcription factor by performing proteomics analysis of the wild-type (WT) and ΔlaeA mutant growing on corn. Notably, our proteomics profile confirmed the functions of extracellular hydrolases, conidial hydrophobin, and response to oxidative stress during the induction of aflatoxin biosynthesis regulated by laeA. Unexpectedly, deletion of laeA resulted in the significant upregulation of the NAD+-dependent histone deacetylase sirA involved in silencing SM clusters via chromatin remodeling. Accompanying the chromatin modification, enzymes participating in SM, including aflatoxin and cyclopiazonic acid biosynthesis, were drastically decreased. Another unexpected finding was that enzymes in the recently identified ustiloxin B biosynthesis pathway might be regulated by laeA. These data provided novel insights into the complex regulation of laeA and suggested a potential link between laeA deletion, NAD+-dependent histone deacetylation, and SM production in A. flavus.


Assuntos
Aspergillus flavus/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Metiltransferases/biossíntese , Metiltransferases/genética , Fatores de Transcrição/biossíntese , Aflatoxinas/biossíntese , Aspergillus flavus/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Histona Desacetilases/biossíntese , Hidrolases/genética , Hidrolases/metabolismo , Indóis/metabolismo , Estresse Oxidativo/fisiologia , Peptídeos Cíclicos/biossíntese , Proteômica , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , Zea mays/microbiologia
7.
Funct Integr Genomics ; 18(5): 533-543, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29730772

RESUMO

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Óleos Industriais/análise , Poluentes do Solo/metabolismo , Bacillus subtilis/classificação , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Bacteriocinas/genética , Biodegradação Ambiental , Biologia Computacional , Mapeamento de Sequências Contíguas , Ontologia Genética , Lipopeptídeos/biossíntese , Lipopeptídeos/genética , Anotação de Sequência Molecular , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Filogenia , Solo/química , Microbiologia do Solo , Tensoativos/química , Tensoativos/metabolismo , Sequenciamento Completo do Genoma
8.
J Am Chem Soc ; 140(19): 6044-6048, 2018 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-29701961

RESUMO

Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules.


Assuntos
Geraniltranstransferase/metabolismo , Peptídeos Cíclicos/biossíntese , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Cianobactérias/química , Cianobactérias/metabolismo , Geraniltranstransferase/isolamento & purificação , Peptídeos Cíclicos/química
9.
J Microbiol Biotechnol ; 28(6): 1030-1036, 2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29642284

RESUMO

Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.


Assuntos
Antifúngicos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Ácidos Graxos/biossíntese , Engenharia Metabólica/métodos , Oligopeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , Mutação Puntual , Antifúngicos/farmacologia , Ácidos Graxos/farmacologia , Perfilação da Expressão Gênica , Oligopeptídeos/farmacologia , Óperon , Peptídeos Cíclicos/farmacologia , Pythium/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Deleção de Sequência
10.
Bioorg Med Chem ; 26(6): 1135-1150, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29295762

RESUMO

Cyclic peptides and cyclotides are becoming common identities within the present efforts seen in peptide engineering - as we seek approaches to achieve potent biological activity, pharmacological selectivity, structurally stability and oral bioavailability. Yet this unique family of peptides has faced uncommon hurdles in their discovery, synthesis and bioengineering, retaining to characteristics that truly deviate these from their linear counterparts. In this mini-review we take a board spectrum approach to introduce this novel family of biomolecules and the troubles that they face in their sequence and disulfide connectivity assignment, together highlighting the present combined strategies involved in cyclic peptide/cyclotide synthesis and modification. These efforts have circumvented otherwise impossible hurdles in their manipulation and production that are only now advancing cyclic peptides/cyclotides as research probes and future pharmaceutical templates.


Assuntos
Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Ciclização , Dissulfetos/química , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/síntese química , Plantas/metabolismo , Ribossomos/metabolismo
11.
BMC Genomics ; 19(1): 45, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29334896

RESUMO

BACKGROUND: Among naturally occurring small molecules, tRNA-derived cyclodipeptides are a class that have attracted attention for their diverse and desirable biological activities. However, no tools are available to link cyclodipeptide synthases identified within prokaryotic genome sequences to their chemical products. Consequently, it is unclear how many genetically encoded cyclodipeptides represent novel products, and which producing organisms should be targeted for discovery. RESULTS: We developed a pipeline for identification and classification of cyclodipeptide biosynthetic gene clusters and prediction of aminoacyl-tRNA substrates and complete chemical structures. We leveraged this tool to conduct a global analysis of tRNA-derived cyclodipeptide biosynthesis in 93,107 prokaryotic genomes, and compared predicted cyclodipeptides to known cyclodipeptide synthase products and all known chemically characterized cyclodipeptides. By integrating predicted chemical structures and gene cluster architectures, we created a unified map of known and unknown genetically encoded cyclodipeptides. CONCLUSIONS: Our analysis suggests that sizeable regions of the chemical space encoded within sequenced prokaryotic genomes remain unexplored. Our map of the landscape of genetically encoded cyclodipeptides provides candidates for targeted discovery of novel compounds. The integration of our pipeline into a user-friendly web application provides a resource for further discovery of cyclodipeptides in newly sequenced prokaryotic genomes.


Assuntos
Bactérias/genética , Dipeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , RNA de Transferência/metabolismo , Algoritmos , Genômica , Fases de Leitura Aberta
12.
ACS Synth Biol ; 7(1): 145-152, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-28866879

RESUMO

Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35mer precursor peptides, which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7-16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including d-Ala, ß-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when l-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.


Assuntos
Peptídeos/metabolismo , Serina Endopeptidases/metabolismo , Agaricales/enzimologia , Agaricales/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/metabolismo , Ciclização , Escherichia coli/metabolismo , Mutagênese Sítio-Dirigida , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/síntese química , Faloidina/química , Faloidina/metabolismo , Serina Endopeptidases/genética , Especificidade por Substrato
13.
J Am Chem Soc ; 139(51): 18623-18631, 2017 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-29190095

RESUMO

The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5'-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5'-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the ß-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.


Assuntos
Metiltransferases/classificação , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Tiazóis/metabolismo , Antibacterianos/biossíntese , Desoxiadenosinas/metabolismo , Hidrogênio/metabolismo , Metilação , Peptídeos Cíclicos/biossíntese , Prótons , S-Adenosil-Homocisteína/metabolismo , Solventes/química
14.
PLoS One ; 12(12): e0188179, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29267290

RESUMO

Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A-F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.


Assuntos
Bacillus subtilis/genética , Genes Bacterianos , Peptídeos Cíclicos/biossíntese , Bacillus subtilis/enzimologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Espectrometria de Massas , Fases de Leitura Aberta
15.
Proc Natl Acad Sci U S A ; 114(49): 12928-12933, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158402

RESUMO

The [4+2] cycloaddition reaction is an enabling transformation in modern synthetic organic chemistry, but there are only limited examples of dedicated natural enzymes that can catalyze this transformation. Thiopeptides (or more formally thiazolyl peptides) are a class of thiazole-containing, highly modified, macrocyclic secondary metabolites made from ribosomally synthesized precursor peptides. The characteristic feature of these natural products is a six-membered nitrogenous heterocycle that is assembled via a formal [4+2] cycloaddition between two dehydroalanine (Dha) residues. This heteroannulation is entirely contingent on enzyme activity, although the mechanism of the requisite pyridine/dehydropiperidine synthase remains to be elucidated. The unusual aza-cylic product is distinct from the more common carbocyclic products of synthetic and biosynthetic [4+2] cycloaddition reactions. To elucidate the mechanism of cycloaddition, we have determined atomic resolution structures of the pyridine synthases involved in the biosynthesis of the thiopeptides thiomuracin (TbtD) and GE2270A (PbtD), in complex with substrates and product analogs. Structure-guided biochemical, mutational, computational, and binding studies elucidate active-site features that explain how orthologs can generate rigid macrocyclic scaffolds of different sizes. Notably, the pyridine synthases show structural similarity to the elimination domain of lanthipeptide dehydratases, wherein insertions of secondary structural elements result in the formation of a distinct active site that catalyzes different chemistry. Comparative analysis identifies other catalysts that contain a shared core protein fold but whose active sites are located in entirely different regions, illustrating a principle predicted from efforts in de novo protein design.


Assuntos
Proteínas de Bactérias/química , Peptídeo Sintases/química , Actinobacteria/enzimologia , Sequência de Aminoácidos , Antibiose , Sítios de Ligação , Biocatálise , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Reação de Cicloadição , Modelos Moleculares , Peptídeos Cíclicos/biossíntese , Ligação Proteica , Tiazóis
16.
Molecules ; 22(10)2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-29065531

RESUMO

Cyclodipeptides (CDP) represent a diverse family of small, highly stable, cyclic peptides that are produced as secondary functional metabolites or side products of protein metabolism by bacteria, fungi, and animals. They are widespread in nature, and exhibit a broad variety of biological and pharmacological activities. CDP synthases (CDPSs) and non-ribosomal peptide synthetases (NRPSs) catalyze the biosynthesis of the CDP core structure, which is further modified by tailoring enzymes often associated with CDP biosynthetic gene clusters. In this review, we provide a comprehensive summary of CDP biosynthetic pathways and modifying enzymes. We also discuss the biological properties of some known CDPs and their possible applications in metabolic engineering.


Assuntos
Vias Biossintéticas , Dipeptídeos/biossíntese , Dipeptídeos/farmacologia , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/farmacologia
17.
Biomed Res Int ; 2017: 5893123, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29082251

RESUMO

This work concerns the study of the enhancement of surfactin and fengycin production by B. mojavensis A21 and application of the produced product in diesel biodegradation. The influences of the culture medium and cells immobilization were studied. The highest lipopeptides production was achieved after 72 hours of incubation in a culture medium containing 30 g/L glucose as carbon source and a combination of yeast extract (1 g/L) and glutamic acid (5 g/L) as nitrogen sources with initial pH 7.0 at 30°C and 90% volumetric aeration. The study of primary metabolites production showed mainly the production of acetoin, with a maximum production after 24 h of strain growth. The use of immobilized cells seemed to be a promising method for improving lipopeptides productivity. In fact, the synthesis of both lipopeptides, mainly fengycin, was greatly enhanced by the immobilization of A21 cells. An increase of diesel degradation capacity of approximately 20, 27, and 40% in the presence of 0.5, 1, and 2 g/L of produced lipopeptides, respectively, was observed. Considering these properties, B. mojavensis A21 strain producing a lipopeptide mixture, containing both surfactin and fengycin, may be considered as a potential candidate for future use in bioremediation and crop protection.


Assuntos
Bacillus/genética , Biodegradação Ambiental , Lipopeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , Bacillus/química , Bacillus/metabolismo , Meios de Cultura , Gasolina/efeitos adversos , Lipopeptídeos/química , Peptídeos Cíclicos/química , Tensoativos/química
18.
Molecules ; 22(11)2017 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-29077011

RESUMO

The aim of the current study was to describe the role and mechanism of Bacillus amyloliquefaciens Y1 against the root-knot nematode, Meloidogyne incognita, under in vitro and in vivo conditions. Initially, the exposure of the bacterial culture supernatant and crude extract of Y1 to M. incognita significantly inhibited the hatching of eggs and caused the mortality of second-stage juveniles (J2), with these inhibitory effects depending on the length of incubation time and concentration of the treatment. The dipeptide cyclo(d-Pro-l-Leu) was identified in B. amyloliquefaciens culture for the first time using chromatographic techniques and nuclear magnetic resonance (NMR ¹H, 13C, H-H COSY, HSQC, and HMBC) and recognized to have nematocidal activity. Various concentrations of cyclo(d-Pro-l-Leu) were investigated for their effect on the hatching of eggs and J2 mortality. Moreover, the in vivo nematocidal activity of the Y1 strain was investigated by conducting pot experiments in which tomato plants were inoculated with M. incognita. Each and every pot was amended 50 mL of fertilizer media (F), or Y1 culture, or nematicide (N) (only once), or fertilizer media with N (FN) at 1, 2, 3, 4 and 5 weeks after transplantation. The results of the pot experiments demonstrated the antagonistic effect of B. amyloliquefaciens Y1 against M. incognita as it significantly decreases the count of eggs and galls per root of the tomato plant as well as the population of J2 in the soil. Besides, the investigation into the growth parameters, such as the length of shoot, shoot fresh and dry weights of the tomato plants, showed that they were significantly higher in the Y1 strain Y1-treated plants compared to F-, FN- and N-treated plants. Therefore, the biocontrol repertoire of this bacterium opens a new insight into the applications in crop pest control.


Assuntos
Antinematódeos/farmacologia , Bacillus amyloliquefaciens/metabolismo , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/farmacologia , Tylenchoidea/efeitos dos fármacos , Animais , Antinematódeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/isolamento & purificação
19.
Acc Chem Res ; 50(10): 2569-2576, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28891639

RESUMO

Natural products are significant therapeutic agents and valuable drug leads. This is likely owing to their three-dimensional structural complexity, which enables them to form complex interactions with biological targets. Enzymes from natural product biosynthetic pathways show great potential to generate natural product-like compounds and libraries. Many challenges still remain in biosynthesis, such as how to rationally synthesize small molecules with novel structures and how to generate maximum chemical diversity. In this Account, we describe recent advances from our laboratory in the synthesis of natural product-like libraries using natural biosynthetic machinery. Our work has focused on the pat and tru biosynthetic pathways to patellamides, trunkamide, and related compounds from cyanobacterial symbionts in marine tunicates. These belong to the cyanobactin class of natural products, which are part of the larger group of ribosomally synthesized and post-translationally modified peptides (RiPPs). These results have enabled the synthesis of rationally designed small molecules and libraries covering more than 1 million estimated derivatives. Because the RiPPs are translated on the ribosome and then enzymatically modified, they are highly compatible with recombinant technologies. This is important because it means that the resulting natural products, their derivatives, and wholly new compounds can be synthesized using the tools of genetic engineering. The RiPPs also represent possibly the most widespread group of bioactive natural products, although this is in part because of the broad definition of what constitutes a RiPP. In addition, the underlying ideas may form the basis for broad-substrate biosynthetic pathways beyond the RiPPs. For example, some of the ideas about kinetic ordering of broad substrate pathways may apply to polyketide or nonribosomal peptide biosynthesis as well. While making these products, we have sought to understand what makes biosynthetic pathways plastic and whether there are any rules that might generally apply to plastic biosynthetic pathways. We present three principles of diversity-generating biosynthesis: (1) substrate evolution, in which the substrates change while enzymes remain constant; (2) pairing of recognition sequences on substrates with biosynthetic enzymes; (3) an inverse metabolic flux in comparison to canonical pathways. If these principles are general, they may enable the design of unimagined derivatives using biosynthetic engineering. For example, it is possible to discover substrate evolution directly by examining sequencing data. By shuffling appropriate recognition sequences and biosynthetic enzymes, it has already been possible to make new hybrid products of multiple pathways. While cases so far have been limited, if this is more general, designed synthesis will become routine. Finally, biosynthesis of natural products is regulated in elaborate ways that are just beginning to be understood. If the inverse metabolic flux model is widespread, it potentially informs on what the timing and relative production level of each enzyme in a designer pathway should be in order to optimize the synthesis of new compounds in vivo.


Assuntos
Produtos Biológicos/síntese química , Vias Biossintéticas , Bibliotecas de Moléculas Pequenas/síntese química , Bactérias/enzimologia , Proteínas de Bactérias/química , Enzimas/química , Cinética , Peptídeos Cíclicos/biossíntese , Sequências Repetitivas de Aminoácidos , Especificidade por Substrato
20.
Chembiochem ; 18(22): 2242-2246, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-28914478

RESUMO

Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction.


Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Inteínas , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Espectrometria de Massas , Modelos Moleculares , Nostoc/química , Synechocystis/química
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