RESUMEN
Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid-derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme-engineering approach to improve the binding of acetyl-CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl-CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding-free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be -52.66 ± 0.11 kcal/mol for the wild type, -55.87 ± 0.16 kcal/mol for the S343A mutant, -60.52 ± 0.25 kcal/mol for T347W mutant, and -59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes.
Asunto(s)
Acetil-CoA Carboxilasa , Streptomyces antibioticus , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Streptomyces antibioticus/metabolismo , Acetilcoenzima A/genética , Simulación del Acoplamiento Molecular , Mutación , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismoRESUMEN
Collagenases are proteases able to degrade native and denatured collagen, with broad applications such as leather, food, and pharmaceutical industries. The aim of this research was to purify and characterize a collagenase from Streptomyces antibioticus. In the present work, the coffee ground substrate provided conditions to obtaining high collagenase activity (377.5 U/mL) using anion-exchange DEAE-Sephadex G50 chromatographic protocol. SDS-PAGE revealed the metallo-collagenase with a single band of 41.28 kDa and was able to hydrolyzed type I and type V collagen producing bioactive peptides that delayed the coagulation time. The enzyme activity showed stability across a range of pH (6.0-11) and temperature (30-55 °C) with optima at pHâ¯7.0 and 60 °C, respectively. Activators include Mg+2, Ca+2, Na+, K+, while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (Km of 27.14 mg/mol, Vmax of 714.29 mg/mol/min, Kcat of 79.9 s-1 and Kcat/Km of 2.95 mL/mg/s) and thermodynamic parameters (Ea of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔGE-S of 8.18 kJ/mol and ΔGE-T of -2.64 kJ/mol) were also defined. Coffee grounds showed to be an interesting source to obtaining a collagenase able to produce bioactive peptides with anticoagulant activity.
Asunto(s)
Streptomyces antibioticus , Café , Termodinámica , Colagenasas , Péptidos , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn-1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with â¼730 mU mg-1 protein of specific activity, and the yield was â¼420 mU l-1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn-3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens.
Asunto(s)
Fosfolipasa D , Streptomyces antibioticus , Plasmalógenos/metabolismo , Streptomyces antibioticus/metabolismo , Fosfolipasa D/genética , Fosfolipasa D/química , Escherichia coli/genética , Escherichia coli/metabolismo , SolubilidadRESUMEN
Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural "natural" polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500-514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.
Asunto(s)
Policétidos , Streptomyces antibioticus , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Modelos Moleculares , Mutagénesis , Streptomyces antibioticus/metabolismoRESUMEN
Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L1 and L2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.
Asunto(s)
Antibacterianos/uso terapéutico , Productos Biológicos/uso terapéutico , Dactinomicina/química , Streptomycetaceae/química , Antibacterianos/química , Productos Biológicos/química , Vías Biosintéticas/efectos de los fármacos , Dactinomicina/análogos & derivados , Dactinomicina/uso terapéutico , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/patogenicidad , Humanos , Streptomyces antibioticus/química , Streptomycetaceae/genética , ortoaminobenzoatos/químicaRESUMEN
Nine new (1-3, 5-8, 11, and 12; named filipins VI-XIV) and three known (4, 9, and 10) filipin-type polyene macrolides were isolated from the deep-sea-derived Streptomyces antibioticus OUCT16-23 using a genome-guided strategy coupled with bioassay. Their structures were elucidated based on the extensive MS and NMR spectroscopic analyses together with ECD calculations. In an antifungal assay, compounds 4, 5, and 7-10 showed different degrees of growth inhibition against Candida albicans with minimum inhibitory concentrations (MICs) of 1.56-12.5 µg/mL, by which the alkyl side-chain substitution affecting the activity was preliminarily studied. A biosynthetic pathway to 1-12 in S. antibioticus OUCT16-23 is also proposed.
Asunto(s)
Streptomyces antibioticus , Streptomyces , Antifúngicos/química , Candida albicans , Filipina/metabolismo , Streptomyces/química , Streptomyces antibioticus/químicaRESUMEN
Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.
Asunto(s)
Proteínas Bacterianas/química , Fosfatos/química , Ácidos Fosfatidicos/química , Fosfatidilinositoles/biosíntesis , Fosfolipasa D/química , Streptomyces antibioticus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Fosfatos/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/química , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces antibioticus/química , Especificidad por SustratoRESUMEN
OBJECTIVE: Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus. RESULTS: Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6ß-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L-1 h-1. NMR spectroscopy confirmed the identity of MDCA as the sole product. CONCLUSIONS: The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Ácido Litocólico/química , Streptomyces antibioticus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Clonación Molecular , Ácido Desoxicólico/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Hidroxilación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Streptomyces antibioticus/genéticaRESUMEN
Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.
Asunto(s)
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Salicilatos/metabolismo , Escherichia coli/metabolismo , Microbiología Industrial , Ingeniería Metabólica/métodos , Streptomyces antibioticus/enzimologíaRESUMEN
Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Bacterianas/química , Lisofosfatidilcolinas/química , Fosfolipasa D/química , Streptomyces antibioticus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lisofosfatidilcolinas/genética , Lisofosfatidilcolinas/metabolismo , Mutagénesis Sitio-Dirigida , Mutación Missense , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Streptomyces antibioticus/genética , Especificidad por Sustrato/genéticaRESUMEN
Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.
Asunto(s)
Proteínas Bacterianas/genética , Monofenol Monooxigenasa/genética , Pseudomonas fluorescens/metabolismo , Streptomyces antibioticus/genética , Proteínas Bacterianas/metabolismo , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Monofenol Monooxigenasa/metabolismo , Pseudomonas fluorescens/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces antibioticus/metabolismoRESUMEN
Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes.
Asunto(s)
Dactinomicina/biosíntesis , Péptido Sintasas/biosíntesis , Streptomyces antibioticus/metabolismo , Streptomyces/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Medios de Cultivo/química , Dactinomicina/química , Escherichia coli/genética , Genes Bacterianos/genética , Vectores Genéticos , Glucosa/metabolismo , Redes y Vías Metabólicas/genética , Familia de Multigenes , Péptido Sintasas/genética , Regiones Promotoras Genéticas , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces antibioticus/genética , Streptomyces antibioticus/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
In the present study we reported the antimicrobial activity of actinomycetes isolated from aridic soil sample collected in Karoo, South Africa. Eighty-six actinomycete strains were isolated and purified, out of them thirty-four morphologically different strains were tested for antimicrobial activity. Among 35 isolates, 10 (28.57%) showed both antibacterial and antifungal activity. The ethyl acetate extract of strain KRG-1 showed the strongest antimicrobial activity and therefore was selected for further investigation. The almost complete nucleotide sequence of the 16S rRNA gene as well as distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain KRG-1 led to the identification of Streptomyces antibioticus KRG-1 (GenBank accession number: KX827270). The ethyl acetate extract of KRG-1 was fractionated by HPLC method against the most suppressed bacterium Staphylococcus aureus (Newman). LC//MS analysis led to the identification of the active peak that exhibited UV-VIS maxima at 442 nm and the ESI-HRMS spectrum showing the prominent ion clusters for [M-H2O+H]+ at m/z 635.3109 and for [M+Na]+ at m/z 1269.6148. This information could be assigned to chromopeptide lactone antibiotic - actinomycin. Our results suggest that unexplored soils could be an interesting source for exploring antibacterial secondary metabolites.
Asunto(s)
Suelo , Actinobacteria/clasificación , Dactinomicina/análisis , Espectrometría de Masas/métodos , Streptomyces antibioticus , ARN Ribosómico 16S , Cromatografía Líquida de Alta Presión/métodos , MétodosRESUMEN
BACKGROUND: Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics. RESULTS: Here, we applied immobilization of Streptomyces lividans 66 using alginate as semi-solid matrix. This alginate-mediated micro-encapsulation increased the production of the extracellular enzyme tyrosinase more than three-fold. The increased production was accompanied by extended viability of the mycelium and a dramatic reduction in the release of intracellular proteins into the culture broth. CONCLUSIONS: Our data demonstrate the utility of micro-encapsulation as a powerful technique to achieve higher yields and lower downstream-processing costs of streptomycetes.
Asunto(s)
Biotecnología/métodos , Monofenol Monooxigenasa/metabolismo , Micelio/fisiología , Streptomyces lividans/fisiología , Alginatos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Inmovilizadas/fisiología , Monofenol Monooxigenasa/genética , Streptomyces antibioticus/genética , Streptomyces lividans/crecimiento & desarrolloRESUMEN
X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X0 ) or 4-oxoproline (Acm X2 ) in their ß-pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPLâ 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPLâ 0 and PPLâ 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acmâ X biosynthetic gene cluster of S.â antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPLâ 1 into PPLâ 0 and PPLâ 2 in vivo as well as in situ in permeabilized cell of the transformed E.â coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectinâ B1 into its keto derivative, 4''-oxoavermectinâ B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Dactinomicina/metabolismo , Ferredoxinas/metabolismo , Prolina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Dactinomicina/química , Hidroxilación , Oxidación-Reducción , Prolina/química , Streptomyces antibioticus/enzimología , Especificidad por SustratoRESUMEN
An actinomycete strain (H12-15) isolated from a sea sediment in a mangrove district was identified as Streptomycesantibioticus on the basis of 16S rDNA gene sequence analysis as well as the investigation of its morphological, physiological, and biochemical characteristics. Two novel benzamido nonacyclic dilactones, namely neoantimycins A (1) and B (2), together with the known antimycins A1ab (3a,b), A2a (4), and A9 (5), were isolated from the culture broth of this strain. Compounds 1 and 2 are the first natural modified ATNs with an unusual benzamide unit. The structures of these new compounds, including their absolute configuration, were established on the basis of HRMS, NMR spectroscopic data, and quantum chemical ECD calculations. Their cytotoxicities against human breast adenocarcinoma cell line MCF-7, the human glioblastoma cell line SF-268, and the human lung cancer cell line NCI-H460 were also tested. All compounds exhibited mild cytotoxic activity. However, Compounds 1 and 2 showed no activity against C. albicans at the test concentration of 1 mg/mL via paper disc diffusion, while the known antimycins showed obvious antifungal activity.
Asunto(s)
Benzamidas/química , Compuestos Orgánicos/química , Streptomyces antibioticus/aislamiento & purificación , Benzamidas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Sedimentos Geológicos/microbiología , Humanos , Células MCF-7 , Estructura Molecular , Compuestos Orgánicos/farmacología , Teoría Cuántica , Streptomyces antibioticus/química , Streptomyces antibioticus/crecimiento & desarrolloRESUMEN
Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.
Asunto(s)
Ácido Cítrico/química , Escherichia coli/enzimología , Polirribonucleótido Nucleotidiltransferasa/química , ARN/química , Streptomyces antibioticus/enzimología , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Evolución Biológica , Ácido Cítrico/metabolismo , Clonación Molecular , Biología Computacional , Secuencia Conservada , Escherichia coli/genética , Exosomas/química , Exosomas/enzimología , Expresión Génica , Humanos , Cinética , Mitocondrias/química , Mitocondrias/enzimología , Simulación del Acoplamiento Molecular , Polirribonucleótido Nucleotidiltransferasa/genética , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN/metabolismo , Estabilidad del ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Streptomyces antibioticus/genética , Homología Estructural de Proteína , Especificidad por Sustrato , Sulfolobus solfataricus/genética , TermodinámicaRESUMEN
An actinomycete strain 200-09, isolated from a soil sample collected from the coast of Hawaii, USA, was identified as Streptomyces antibioticus on the basis of its morphological, physiological and biochemical characteristics as well as 16S rDNA analysis. A new antimycin-type antibiotic, kitamycin C (1), together with kitamycin A (2), kitamycin B (3), urauchmycin B (4), deisovaleryblastomycin (5) was isolated from a cultured broth of strain 200-09. The structure of the new compound was determined by spectroscopic data, including HR-ESI-MS and NMR. All the compounds exhibited antifungal activities against Candida albicans with MIC of about 25.0 µg mL-1.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Macrólidos/química , Macrólidos/farmacología , Streptomyces antibioticus/química , Antibacterianos/química , Antifúngicos/química , Antimicina A/análogos & derivados , Antimicina A/farmacología , Candida albicans/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.
Asunto(s)
Antibacterianos/biosíntesis , Inhibidores Enzimáticos/metabolismo , Pentostatina/biosíntesis , Vidarabina/biosíntesis , Adenosina Desaminasa/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Secuencia de Bases , Análisis por Conglomerados , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pentostatina/química , Pentostatina/farmacología , Análisis de Secuencia de ADN , Streptomyces antibioticus/genética , Vidarabina/química , Vidarabina/farmacologíaRESUMEN
Chlorothricin, isolated from Streptomyces antibioticus, is a parent member of spirotetronate family of antibiotics that have long been appreciated for their remarkable biological activities. ChlF1 plays bifunctional roles in chlorothricin biosynthesis by binding to its target genes (chlJ, chlF1, chlG, and chlK). The dissociation constants of ChlF1 to these genes are â¼ 102-140 nm. A consensus sequence, 5'-GTAANNATTTAC-3', was found in these binding sites. ChlF1 represses the transcription of chlF1, chlG, and chlK but activates chlJ, which encodes a key enzyme acyl-CoA carboxyl transferase involved in the chlorothricin biosynthesis. We demonstrate that the end product chlorothricin and likewise its biosynthetic intermediates (demethylsalicycloyl chlorothricin and deschloro-chlorothricin) can act as signaling molecules to modulate the binding of ChlF1 to its target genes. Intriguingly, a correlation between the antibacterial activity and binding ability of signaling molecules to the regulator ChlF1 is clearly observed. These features of the signaling molecules are associated with the glycosylation of spirotetronate macrolide aglycone. The findings provide new insights into the TetR family regulators responding to special structure of signaling molecules, and we reveal the regulatory mini-network mediated by ChlF1 in chlorothricin biosynthesis for the first time.