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1.
Angew Chem Int Ed Engl ; 62(31): e202306429, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37283082

ABSTRACT

A gene coding for a terpene synthase homolog from Kitasatospora viridis was cloned and expressed in Escherichia coli. The purified recombinant protein possessed sesterterpene synthase activity and efficiently converted geranylfarnesyl diphosphate (GFPP) with 19 % yield into the sesterterpene hydrocarbon sesterviridene A. Large scale enzymatic conversions also allowed for the isolation of two side products that are generated with very low yields of ca. 0.1 %. Several derivatives of sesterviridene A were obtained by chemical transformations, securing the NMR-based structural assignments. The absolute configuration of sesterviridene A was determined by chemical correlation using stereoselectively deuterated precursors and by anomalous dispersion X-ray crystallography. The cyclisation mechanism from GFPP to sesterviridene A was extensively studied through isotopic labelling experiments and DFT calculations.


Subject(s)
Alkyl and Aryl Transferases , Streptomycetaceae , Sesterterpenes/chemistry , Streptomycetaceae/metabolism , Recombinant Proteins
2.
J Agric Food Chem ; 71(17): 6584-6593, 2023 May 03.
Article in English | MEDLINE | ID: mdl-37076425

ABSTRACT

The marine-derived Streptomyces sp. FIMYZ-003 strain was found to produce novel siderophores with yields negatively correlated with the iron concentration in the medium. Mass spectrometry (MS)-based metabolomics coupled with metallophore assays identified two novel α-hydroxycarboxylate-type siderophores, fradiamines C and D (3 and 4), together with two related known siderophores, fradiamines A and B (1 and 2). Their chemical structures were elucidated by nuclear magnetic resonance (NMR) and MS experiments. The annotation of a putative fra biosynthetic gene cluster enabled us to propose the biosynthetic pathway of fradiamines A-D. Furthermore, the solution-phase iron-binding activity of fradiamines was evaluated using metabolomics, confirming them as general iron scavengers. Fradiamines A-D exhibited Fe(III) binding activity equivalent to that of deferoxamine B mesylate. Growth analysis of pathogenic microbes demonstrated that fradiamine C promoted the growth of Escherichia coli and Staphylococcus aureus, but fradiamines A, B, and D did not. The results indicate that fradiamine C may serve as a novel iron carrier applicable to antibiotic delivery strategies to treat and prevent foodborne pathogens.


Subject(s)
Ferric Compounds , Siderophores , Chelating Agents , Iron/metabolism , Metabolomics , Siderophores/chemistry , Siderophores/genetics , Siderophores/metabolism , Streptomycetaceae/chemistry , Streptomycetaceae/metabolism
3.
J Environ Sci Health B ; 57(6): 470-478, 2022.
Article in English | MEDLINE | ID: mdl-35438041

ABSTRACT

The residues of imazamox (IMX) will cause phytotoxicity to subsequent crops after long-term use, and will also pollute the soil and its surrounding environment. This study isolates and identifies two strains of Streptomycetaceae JX02 and JX06 that can effectively degrade IMX. Use response surface method Box-Behnken design to optimize physicochemical parameters. The optimal degradation conditions of strains JX02 and JX06 are obtained and verified: IMX concentration is 150 mg L-1, the initial dosage is 9.9%, 9.1% (OD600 = 0.1), the temperature is 26.4 and 27.5 °C, and pH value is 7.0 and 7.7, respectively. The degradation rates of 150 mg L-1 IMX detected by HPLC within 4 d were 99 and 94%, respectively. After adding strains JX02 and JX06, the half-life of IMX in the soil is shortened to 11 d and 13 d, indicating that Streptomycetaceae had a positive effect on the remediation of soil. It is expected to provide scientific information for the rational use, environmental safety evaluation of IMX, and provide a basis for future research and development of microbial agents.


Subject(s)
Soil Pollutants , Streptomycetaceae , Biodegradation, Environmental , Imidazoles , Soil/chemistry , Soil Pollutants/metabolism , Streptomycetaceae/metabolism
4.
Nat Chem Biol ; 17(4): 485-491, 2021 04.
Article in English | MEDLINE | ID: mdl-33462497

ABSTRACT

Tryptophan 2C methyltransferase (TsrM) methylates C2 of the indole ring of L-tryptophan during biosynthesis of the quinaldic acid moiety of thiostrepton. TsrM is annotated as a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5'-deoxyadenosyl 5'-radical intermediate, a hallmark of radical SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae, which are the first structures of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor, and a [4Fe-4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CXXXCXXC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating the formation of a C2 carbanion.


Subject(s)
Methyltransferases/metabolism , Methyltransferases/ultrastructure , Arginine/chemistry , Catalysis , Coenzymes , Iron-Sulfur Proteins/metabolism , Methylation , S-Adenosylmethionine , Streptomycetaceae/genetics , Streptomycetaceae/metabolism , Thiostrepton/biosynthesis , Tryptophan/metabolism , Vitamin B 12/chemistry , X-Ray Diffraction/methods
5.
Mol Microbiol ; 115(6): 1181-1190, 2021 06.
Article in English | MEDLINE | ID: mdl-33278050

ABSTRACT

The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling.


Subject(s)
Cell Wall/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Osmotic Pressure , Streptomycetaceae/metabolism , Anaerobiosis/physiology , Cryoelectron Microscopy , Cytoskeletal Proteins/genetics , Cytoskeleton/genetics , Electron Microscope Tomography , Intermediate Filaments/genetics , Oxygen/metabolism , Soil Microbiology , Streptomycetaceae/genetics
6.
Antonie Van Leeuwenhoek ; 113(6): 825-837, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32060816

ABSTRACT

Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.


Subject(s)
Genome, Bacterial , Protoplasts , Streptomycetaceae/genetics , DNA Transposable Elements , Genetic Heterogeneity , Plasmids , Regeneration , Streptomycetaceae/metabolism
7.
Nucleic Acids Res ; 48(3): 1583-1598, 2020 02 20.
Article in English | MEDLINE | ID: mdl-31956908

ABSTRACT

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.


Subject(s)
Bacterial Proteins/genetics , Bambermycins/biosynthesis , Biological Products/metabolism , Cyclic GMP/analogs & derivatives , DNA-Binding Proteins/genetics , Transcription Factors/genetics , Bacterial Proteins/antagonists & inhibitors , Cyclic GMP/genetics , Cyclic GMP/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Escherichia coli Proteins/genetics , Gene Deletion , Gene Expression Regulation, Bacterial/genetics , Nucleotides/genetics , Peptidoglycan Glycosyltransferase/antagonists & inhibitors , Phosphorus-Oxygen Lyases/genetics , Second Messenger Systems/genetics , Streptomycetaceae/genetics , Streptomycetaceae/metabolism , Transcription Factors/antagonists & inhibitors
8.
J Antibiot (Tokyo) ; 73(3): 167-170, 2020 03.
Article in English | MEDLINE | ID: mdl-31735911

ABSTRACT

Small cell lung cancer (SCLC) is a severe malignancy with early and widespread metastasis, and novel therapeutic drugs are needed. To identify cytotoxic natural compounds against SCLC, we screened libraries of microbial fermentation broths using several lung cancer cell lines. We found that the actinomycete strain MG372-hF19 produces a compound that has not been isolated from natural sources but previously chemically synthesized, 6-chloro-1H-indole-3-carboxaldehyde (1), and an entirely new compound, named 6-deoxy-α-L-talopyranose 1-(6-chloro-1H-indole-3-carboxylate) (2), together with leptomycins. The molecular formulas of the compounds were established as C9H6ClNO and C15H16ClNO6, respectively, via high-resolution electrospray ionization mass spectrometry, and their structures were determined using detailed NMR. Absolute configurational analysis of the sugar unit of compound 2 revealed that the compound incorporates the rare deoxyhexose 6-deoxy-α-L-talopyranose. Both compounds exhibited weak growth-inhibiting activities against human lung cancer cell lines.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Glycosides/chemistry , Streptomycetaceae/metabolism , Antineoplastic Agents/metabolism , Cell Line, Tumor , Humans , Small Cell Lung Carcinoma/drug therapy
9.
Appl Environ Microbiol ; 85(13)2019 07 01.
Article in English | MEDLINE | ID: mdl-31028028

ABSTRACT

Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.


Subject(s)
Bacterial Proteins/genetics , Carbohydrate Dehydrogenases/genetics , Peroxidases/genetics , Streptomycetaceae/genetics , Bacterial Proteins/metabolism , Carbohydrate Dehydrogenases/metabolism , Oxidation-Reduction , Oxidoreductases/metabolism , Peroxidases/metabolism , Streptomycetaceae/enzymology , Streptomycetaceae/metabolism
10.
Nat Chem Biol ; 15(2): 111-114, 2019 02.
Article in English | MEDLINE | ID: mdl-30598544

ABSTRACT

Here we report a transcription factor decoy strategy for targeted activation of eight large silent polyketide synthase and non-ribosomal peptide synthetase gene clusters, ranging from 50 to 134 kilobases (kb) in multiple streptomycetes, and characterization of a novel oxazole family compound produced by a 98-kb biosynthetic gene cluster. Owing to its simplicity and ease of use, this strategy can be scaled up readily for discovery of natural products in streptomycetes.


Subject(s)
Peptide Synthases/genetics , Polyketide Synthases/genetics , Transcription Factors/biosynthesis , Gene Expression Regulation/genetics , Multigene Family/physiology , Peptide Synthases/physiology , Polyketide Synthases/physiology , Streptomycetaceae/metabolism
11.
Mol Biol Rep ; 45(6): 2563-2570, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30311126

ABSTRACT

The search for new compounds effective against Mycobacterium tuberculosis is still a priority in medicine. The evaluation of microorganisms isolated from non-conventional locations offers an alternative to look for new compounds with antimicrobial activity. Endophytes have been successfully explored as source of bioactive compounds. In the present work we studied the nature and antimycobacterial activity of a compound produced by Streptomyces scabrisporus, an endophyte isolated from the medicinal plant Amphipterygium adstringens. The active compound was detected as the main secondary metabolite present in organic extracts of the streptomycete and identified by NMR spectroscopic data as steffimycin B (StefB). This anthracycline displayed a good activity against M. tuberculosis H37Rv ATCC 27294 strain, with MIC100 and SI values of 7.8 µg/mL and 6.42, respectively. When tested against the rifampin mono resistant M. tuberculosis Mtb-209 pathogen strain, a better activity was observed (MIC100 of 3.9 µg/mL), suggesting a different action mechanism of StefB from that of rifampin. Our results supported the endophyte Streptomyces scabrisporus as a good source of StefB for tuberculosis treatment, as this anthracycline displayed a strong bactericidal effect against M. tuberculosis, one of the oldest and more dangerous human pathogens causing human mortality.


Subject(s)
Anthracyclines/pharmacology , Sapindaceae/metabolism , Anacardiaceae , Anthracyclines/isolation & purification , Anthracyclines/metabolism , Anti-Infective Agents/pharmacology , Antitubercular Agents , Endophytes/isolation & purification , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Plant Extracts/isolation & purification , Plants, Medicinal/metabolism , Sapindaceae/toxicity , Streptomycetaceae/metabolism
12.
J Antibiot (Tokyo) ; 71(10): 854-861, 2018 10.
Article in English | MEDLINE | ID: mdl-29973681

ABSTRACT

ß-Carboline alkaloids and related compounds show a broad spectrum of biological activities. We previously identified new members of the ß-carboline alkaloid family by using an engineered Kitasatospora setae strain and a heterologous Streptomyces host expressing the plausible biosynthetic genes, including the hypothetical gene kse_70640 (kslB). Here, we elucidated the chemical structure of a new tetrahydro-ß-carboline compound (named kitasetalic acid) that appeared in a heterologous Streptomyces host expressing the kslB gene alone. Kitasetalic acid suppressed the expression of glucose-regulated protein 78 (GRP78) without inducing cell death. This is the first report to show that a tetrahydro-ß-carboline compound regulates the expression of the GRP78 protein in cancer cell lines.


Subject(s)
Carbolines/metabolism , Genetic Engineering , Glucose/pharmacology , Streptomycetaceae/metabolism , Carbolines/chemistry , Carbolines/pharmacology , Cell Line , Endoplasmic Reticulum Chaperone BiP , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation/drug effects , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Molecular Structure , Streptomycetaceae/genetics
13.
J Microbiol ; 56(8): 571-578, 2018 Aug.
Article in English | MEDLINE | ID: mdl-30047086

ABSTRACT

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.


Subject(s)
5-Aminolevulinate Synthetase/metabolism , Biosynthetic Pathways/genetics , Coenzyme A Ligases/metabolism , Cyclopentanes/metabolism , Streptomycetaceae/enzymology , Streptomycetaceae/metabolism , 5-Aminolevulinate Synthetase/genetics , Cloning, Molecular , Coenzyme A Ligases/genetics , Coenzymes/analysis , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors , Hydrogen-Ion Concentration , Plasmids , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Streptomycetaceae/genetics
14.
J Microbiol Immunol Infect ; 51(1): 45-54, 2018 Feb.
Article in English | MEDLINE | ID: mdl-27103501

ABSTRACT

BACKGROUND/PURPOSE: In this study, an acidophilic actinobacteria strain was used as a novel reducing agent for a single-step synthesis of nanostructure silver particles. We used a Streptacidiphilus durhamensis HGG16n isolate for efficient synthesis of bioactive silver nanoparticles [bio(AgNPs)] in an inexpensive, eco-friendly, and nontoxic manner. The obtained bio(AgNPs) exhibited unique physicochemical and biochemical properties. METHODS: Structural, morphological, and optical properties of the synthesized biocolloids were characterized by spectroscopy, dynamic light scattering, and electron microscopy approaches. The antimicrobial activity was evaluated using the well- and disc-diffusion methods. RESULTS: The obtained crystalline structure and stable biosynthesized silver nanoparticles ranged in size from 8 nm to 48 nm and were mostly spherical in shape. Antimicrobial assays of the silver nanoparticles against pathogenic bacteria showed the highest antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus mirabilis, followed by Escherichia coli, Klebsiella pneumoniae, and Bacillus subtilis. Moreover, the synergistic effect of bio(AgNPs) with various commercially available antibiotics was also evaluated. CONCLUSION: These results provide insight into the development of new antimicrobial agents along with synergistic enhancement of the antibacterial mechanism against clinical bacteria.


Subject(s)
Anti-Infective Agents/metabolism , Anti-Infective Agents/pharmacology , Metal Nanoparticles/chemistry , Silver/metabolism , Silver/pharmacology , Streptomycetaceae/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Escherichia coli/drug effects , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Proteus mirabilis/drug effects , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects
15.
Biotechnol J ; 13(1)2018 Jan.
Article in English | MEDLINE | ID: mdl-29076639

ABSTRACT

Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism. This new trend has been accompanied with development of new computational resources (genome and compound mining tools), generation of various high-quality omics data, establishment of molecular tools, and other strain engineering strategies. They all come together to enable systems metabolic engineering of streptomycetes, allowing more systematic and efficient strain development. In this review, the authors present recent progresses within systems metabolic engineering of streptomycetes for uncovering their hidden potential to produce novel compounds and for the improved production of secondary metabolites.


Subject(s)
Metabolic Engineering/trends , Streptomycetaceae/metabolism , Systems Biology , Secondary Metabolism/genetics , Streptomycetaceae/genetics
16.
Angew Chem Int Ed Engl ; 56(23): 6665-6668, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28452105

ABSTRACT

Belactosins and cystargolides are natural product proteasome inhibitors from Actinobacteria. Both feature dipeptidic backbones and a unique ß-lactone building block. Herein, we present a detailed investigation of their biosynthesis. Identification and analysis of the corresponding gene clusters indicated that both compounds are assembled by rare single-enzyme amino acid ligases. Feeding experiments with isotope-labeled precursors and in vitro biochemistry showed that the formation of the ß-lactone warhead is unprecedented and reminiscent of leucine biosynthesis, and that it involves the action of isopropylmalate synthase homologues.


Subject(s)
Dipeptides/metabolism , Lactones/chemistry , Peptides/metabolism , Proteasome Inhibitors/chemical synthesis , Streptomycetaceae/metabolism , Amino Acids/metabolism , Genome, Bacterial , Intercellular Signaling Peptides and Proteins , Ligases/genetics , Ligases/metabolism , Magnetic Resonance Spectroscopy , Multigene Family , Streptomycetaceae/genetics , Tandem Mass Spectrometry
17.
J Antibiot (Tokyo) ; 70(5): 506-513, 2017 May.
Article in English | MEDLINE | ID: mdl-28196972

ABSTRACT

The genus Kitasatospora was proposed in 1982. Although Kitasatospora strains resemble Streptomyces strains in morphology, they are clearly different in cell-wall composition, as they contain both LL- and meso-diaminopimelic acid. Aerial and submerged spores contain LL-, while vegetative and submerged mycelia contain mainly meso- in their cell walls. Currently, 23 species have been validly proposed. Members of the genus Kitasatospora form a tight cluster and represent a legitimate genus distinct from Streptomyces on the basis of phylogenetic analysis of 16S rRNA gene sequences. A variety of biologically active compounds have been found from Kitasatospora strains and structures of these compounds are extremely diverse. Genome sequences of 15 strains published so far are about 7-9 Mb in size and contain many genes governing secondary metabolites.


Subject(s)
RNA, Ribosomal, 16S/genetics , Secondary Metabolism/genetics , Streptomycetaceae/genetics , Cell Wall/chemistry , Genome, Bacterial , Phylogeny , Sequence Analysis, RNA , Species Specificity , Streptomyces/classification , Streptomyces/genetics , Streptomycetaceae/classification , Streptomycetaceae/metabolism
18.
J Antibiot (Tokyo) ; 70(5): 542-550, 2017 May.
Article in English | MEDLINE | ID: mdl-28196975

ABSTRACT

Tyropeptins are new proteasome inhibitors isolated from the culture broth of Kitasatospora sp. MK993-dF2. Tyropeptins permeate cell membranes, inhibit intracellular proteasomes and reduce the degradation of ubiquitinated proteins in mammalian cells. We performed structure-based drug design and structure-activity relationship studies on tyropeptin derivatives to obtain valuable information of derivatives. Among the synthesized tyropeptin derivatives, some boronic acid derivatives exhibited potent antitumor effects against human multiple myeloma. In this review, we summarize the discovery of tyropeptins and the development of tyropeptin derivatives.


Subject(s)
Dipeptides/isolation & purification , Drug Design , Streptomycetaceae/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Dipeptides/chemistry , Dipeptides/pharmacology , Humans , Proteasome Endopeptidase Complex , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/isolation & purification , Proteasome Inhibitors/pharmacology , Structure-Activity Relationship
19.
Biochemistry ; 55(23): 3270-84, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27221142

ABSTRACT

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known. Using a combination of enzymolysis and mass spectrometry, we compared the properties of α-glucan isolated from actinomycetes with polymer synthesized in vitro by GlgE and GlgB. We now propose the following assembly mechanism. Polymer synthesis starts with GlgE and its donor substrate, α-maltose 1-phosphate, yielding a linear oligomer with a degree of polymerization (∼16) sufficient for GlgB to introduce a branch. Branching involves strictly intrachain transfer to generate a C chain (the only constituent chain to retain its reducing end), which now bears an A chain (a nonreducing end terminal branch that does not itself bear a branch). GlgE preferentially extends A chains allowing GlgB to act iteratively to generate new A chains emanating from B chains (nonterminal branches that themselves bear a branch). Although extension and branching occur primarily with A chains, the other chain types are sometimes extended and branched such that some B chains (and possibly C chains) bear more than one branch. This occurs less frequently in α-glucans than in classical glycogens. The very similar properties of cytosolic and capsular α-glucans from Mycobacterium tuberculosis imply GlgE and GlgB are sufficient to synthesize them both.


Subject(s)
Glucans/chemistry , Glucans/metabolism , Glucosyltransferases/metabolism , Mycobacterium/metabolism , Streptomycetaceae/metabolism , Sugar Phosphates/metabolism , Electrophoresis, Capillary , Magnetic Resonance Spectroscopy , Mycobacterium/classification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
20.
ACS Chem Biol ; 11(2): 478-90, 2016 Feb 19.
Article in English | MEDLINE | ID: mdl-26675041

ABSTRACT

The increasing bacterial multidrug resistance necessitates novel drug-discovery efforts. One way to obtain novel chemistry is glycosylation, which is prevalent in nature, with high diversity in both the sugar moieties and the targeted aglycones. Kitasatospora sp. MBT66 produces endophenaside antibiotics, which is a family of (methyl-)rhamnosylated phenazines. Here we show that this strain also produces the plecomacrolide leucanicidin (1), which is derived from bafilomycin A1 by glycosylation with the same methyl-rhamnosyl moiety as present in the endophenasides. Immediately adjacent to the baf genes for bafilomycin biosynthesis lie leuA and leuB, which encode a sugar-O-methyltransferase and a glycosyltransferase, respectively. LeuA and LeuB are the only enzymes encoded by the genome of Kitasatospora sp. MBT66 that are candidates for the methyl-rhamnosylation of natural products, and mutation of leuB abolished glycosylation of both families of natural products. Thus, LeuA and -B mediate the post-PKS methyl-rhamnosylation of bafilomycin A1 to leucanicidin and of phenazines to endophenasides, showing surprising promiscuity by tolerating both macrolide and phenazine skeletons as the substrates. Detailed metabolic analysis by MS/MS based molecular networking facilitated the characterization of nine novel phenazine glycosides 6-8, 16, and 22-26, whereby compounds 23 and 24 represent an unprecedented tautomeric glyceride phenazine, further enriching the structural diversity of endophenasides.


Subject(s)
Anti-Bacterial Agents/metabolism , Glycosyltransferases/metabolism , Macrolides/metabolism , Methyltransferases/metabolism , Phenazines/metabolism , Streptomycetaceae/enzymology , Anti-Bacterial Agents/chemistry , Glycosyltransferases/genetics , Macrolides/chemistry , Metabolic Networks and Pathways , Methyltransferases/genetics , Multigene Family , Mutation , Phenazines/chemistry , Streptomycetaceae/chemistry , Streptomycetaceae/genetics , Streptomycetaceae/metabolism , Substrate Specificity , Tandem Mass Spectrometry
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