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1.
J Nat Prod ; 87(4): 831-836, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38551509

RESUMEN

Two novel polyketides, accraspiroketides A (1) and B (2), which feature unprecedented [6 + 6+6 + 6] + [5 + 5] spiro chemical architectures, were isolated from Streptomyces sp. MA37 ΔaccJ mutant strain. Compounds 1-2 exhibit excellent activity against Gram-positive bacteria (MIC = 1.5-6.3 µg/mL). Notably, 1 and 2 have superior activity against clinically isolated Enterococcus faecium K60-39 (MIC = 4.0 µg/mL and 4.7 µg/mL, respectively) than ampicillin (MIC = 25 µg/mL).


Asunto(s)
Antibacterianos , Enterococcus faecium , Pruebas de Sensibilidad Microbiana , Policétidos , Streptomyces , Policétidos/farmacología , Policétidos/química , Policétidos/aislamiento & purificación , Streptomyces/química , Estructura Molecular , Antibacterianos/farmacología , Antibacterianos/química , Enterococcus faecium/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Compuestos de Espiro/aislamiento & purificación , Naftacenos/química , Naftacenos/farmacología
2.
Molecules ; 28(1)2023 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-36615552

RESUMEN

Indole-containing acyloins are either key intermediates of many antimicrobial/antiviral natural products or building blocks in the synthesis of biologically active molecules. As such, access to structurally diverse indole-containing acyloins has attracted considerable attention. In this report, we present a pilot study of using biotransformation to provide acyloins that contain various indole substituents. The biotransformation system contains the tryptophan synthase standalone ß-subunit variant, PfTrpB6, generated from directed evolution in the literature; a commercially available L-amino acid oxidase (LAAO); and the thiamine-diphosphate (ThDP)-dependent enzyme NzsH, encoded in the biosynthetic gene cluster (nzs) of the bacterial carbazole alkaloid natural product named neocarazostatin A. The utilization of the first two enzymes, the PfTrpB variant and LAAO, is designed to provide structurally diverse indole 3-pyruvate derivatives as donor substrates for NzsH-catalysed biotransformation to provide acyloin derivatives. Our results demonstrate that NzsH displays a considerable substrate profile toward donor substrates for production of acyloins with different indole ring systems, suggesting that NzsH could be further explored as a potential biocatalyst via directed evolution to improve the catalytic efficiency in the future.


Asunto(s)
Alcoholes Grasos , Indoles , Proyectos Piloto , Indoles/química , Alcoholes Grasos/química , Ácido Pirúvico
3.
Beilstein J Org Chem ; 18: 1763-1771, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36632531

RESUMEN

During the continued isolation of different bacteria from highly diverse, low human activity environments in Ghana and the subsequent characterization and biological activity studies of their secondary metabolites, we found both Gram-positive and Gram-negative Bacillus strains to be ubiquitous and widespread. One of such strains, the Ghanaian novel Bacillus sp. strain DE2B was isolated from rhizosphere soils collected from the Digya National Park in Ghana. Chromatographic purifications of the fermented culture extract of the strain DE2B, led to the isolation of a cyclic lipopeptide, digyalipopeptide A (1). Using 1D and 2D NMR data, mass spectrometry sequence tagging, advanced Marfey's analysis, and the GNPS molecular networking we solved the full structure of digyalipopeptide A (1). We found that compound 1 is a member of a somewhat homologous series of peptides produced as a mixture by the strain containing the same amino acid sequence in the cyclic peptide backbone but differing only by the length of aliphatic fatty acid side chains. When tested against Trypanosoma brucei subsp. brucei strain GUTat 3.1 and Leishmania donovani (Laveran and Mesnil) Ross (D10), digyalipopeptide A (1) gave IC50 values of 12.89 µM (suramin IC50 0.96 µM) and 4.85 µM (amphotericin B IC50 4.87 µM), respectively. Furthermore, digyalipopeptide A (1) produced IC50 values of 10.07 µM (ampicillin IC50 0.18 µM) and 10.01 µM (ampicillin IC50 1.53 µM) for Staphylococcus aureus and Shigella sonnei, respectively. The selectivity and toxicity profile of compound 1 was investigated using normal cell lines, macrophages RAW 264.7. When tested against normal macrophages, compound 1 gave an IC50 value of 71.32 µM. Selectivity indices (SI) were obtained by calculating the ratio of the IC50 in RAW 264.7 to the IC50 in the respective microbe and neglected parasite. In the presence of RAW 264.7 cell lines, compound 1 was particularly selective towards Leishmania donovani (Laveran and Mesnil) Ross (D10) with an SI value of 14.71. The bioactivity studies conducted confirm the role of these cyclic lipopeptides as defense chemicals in their natural environment and their ability to be biologically active across different species.

4.
Mar Drugs ; 19(6)2021 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-34205180

RESUMEN

Three dermacozines, dermacozines N-P (1-3), were isolated from the piezotolerant Actinomycete strain Dermacoccus abyssi MT 1.1T, which was isolated from a Mariana Trench sediment in 2006. Herein, we report the elucidation of their structures using a combination of 1D/2D NMR, LC-HRESI-MSn, UV-Visible, and IR spectroscopy. Further confirmation of the structures was achieved through the analysis of data from density functional theory (DFT)-UV-Visible spectral calculations and statistical analysis such as two tailed t-test, linear regression-, and multiple linear regression analysis applied to either solely experimental or to experimental and calculated 13C-NMR chemical shift data. Dermacozine N (1) bears a novel linear pentacyclic phenoxazine framework that has never been reported as a natural product. Dermacozine O (2) is a constitutional isomer of the known dermacozine F while dermacozine P (3) is 8-benzoyl-6-carbamoylphenazine-1-carboxylic acid. Dermacozine N (1) is unique among phenoxazines due to its near infrared (NIR) absorption maxima, which would make this compound an excellent candidate for research in biosensing chemistry, photodynamic therapy (PDT), opto-electronic applications, and metabolic mapping at the cellular level. Furthermore, dermacozine N (1) possesses weak cytotoxic activity against melanoma (A2058) and hepatocellular carcinoma cells (HepG2) with IC50 values of 51 and 38 µM, respectively.


Asunto(s)
Actinobacteria/química , Sedimentos Geológicos/microbiología , Fenazinas/química , Fenazinas/aislamiento & purificación , Procesos Fotoquímicos , Luz , Espectroscopía de Resonancia Magnética , Análisis de Regresión , Espectrofotometría/métodos
5.
Molecules ; 26(15)2021 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-34361641

RESUMEN

The search for novel antitrypanosomals and the investigation into their mode of action remain crucial due to the toxicity and resistance of commercially available antitrypanosomal drugs. In this study, two novel antitrypanosomals, tortodofuordioxamide (compound 2) and tortodofuorpyramide (compound 3), were chemically derived from the natural N-alkylamide tortozanthoxylamide (compound 1) through structural modification. The chemical structures of these compounds were confirmed through spectrometric and spectroscopic analysis, and their in vitro efficacy and possible mechanisms of action were, subsequently, investigated in Trypanosoma brucei (T. brucei), one of the causative species of African trypanosomiasis (AT). The novel compounds 2 and 3 displayed significant antitrypanosomal potencies in terms of half-maximal effective concentrations (EC50) and selectivity indices (SI) (compound 1, EC50 = 7.3 µM, SI = 29.5; compound 2, EC50 = 3.2 µM, SI = 91.3; compound 3, EC50 = 4.5 µM, SI = 69.9). Microscopic analysis indicated that at the EC50 values, the compounds resulted in the coiling and clumping of parasite subpopulations without significantly affecting the normal ratio of nuclei to kinetoplasts. In contrast to the animal antitrypanosomal drug diminazene, compounds 1, 2 and 3 exhibited antioxidant absorbance properties comparable to the standard antioxidant Trolox (Trolox, 0.11 A; diminazene, 0.50 A; compound 1, 0.10 A; compound 2, 0.09 A; compound 3, 0.11 A). The analysis of growth kinetics suggested that the compounds exhibited a relatively gradual but consistent growth inhibition of T. brucei at different concentrations. The results suggest that further pharmacological optimization of compounds 2 and 3 may facilitate their development into novel AT chemotherapy.


Asunto(s)
Tripanocidas , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Ratones , Células RAW 264.7 , Tripanocidas/síntesis química , Tripanocidas/química , Tripanocidas/farmacología , Tripanosomiasis Africana/metabolismo
6.
Org Biomol Chem ; 18(12): 2219-2222, 2020 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-32159577

RESUMEN

More than 500 siderophores are known to date, but only three were identified to be aryl-containing hydroxamate siderophores, legonoxamines A and B from Streptomyces sp. MA37, and aryl ferrioxamine 2 from Micrococcus luteus KLE1011. Siderophores are produced by microorganisms to scavenge iron from the environment, thereby making this essential metal nutrient available to the microbe. We demonstrate here that LgoC from MA37 is responsible for the key aryl-hydroxamate forming step in legonoxamine biosynthesis. Biochemical characterization established that LgoC displays considerable promiscuity for the acylation between N-hydroxy-cadaverine and SNAC (N-acetylcysteamines) thioester derivatives.


Asunto(s)
Coenzima A Transferasas/metabolismo , Sideróforos/metabolismo , Acilación , Proteínas Bacterianas/metabolismo , Ácidos Hidroxámicos/química , Hierro/metabolismo , Micrococcus luteus/química , Sideróforos/biosíntesis , Sideróforos/aislamiento & purificación , Streptomyces/química , Streptomyces/enzimología
7.
Appl Microbiol Biotechnol ; 104(9): 3885-3896, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-32140842

RESUMEN

ß-Hydroxy-α-amino acids (ßH-AAs) are key components of many bioactive molecules as well as exist as specialised metabolites. Among these ßH-AAs, 4-fluorothreonine (4-FT) is the only naturally occurring fluorinated AA discovered thus far. Here we report overexpression and biochemical characterisation of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA), a homologue of FTase previously identified in the biosynthesis of 4-FT in S. cattleya. FTaseMA displays considerable substrate plasticity to generate 4-FT as well as other ß-hydroxy-α-amino acids with various functionalities at C4 position, giving the prospect of new chemo-enzymatic applications. The enzyme has a hybrid of two catalytic domains, serine hydroxymethyltransferase (S) and aldolase (A). Site-directed mutagenesis allowed the identification of the key residues of FTases, suggesting that the active site of A domain has a historical reminiscent feature in metal-dependent aldolases. Elemental analysis demonstrated that FTaseMA is indeed a Zn2+-dependent enzyme, the first example of pyridoxal phosphate (PLP) enzyme family fused with a metal-binding domain carrying out a distinct catalytic role. Finally, FTaseMA showed divergent evolutionary origin with other PLP dependent enzymes.


Asunto(s)
Aminoácidos Aromáticos/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Treonina/análogos & derivados , Transaldolasa/metabolismo , Zinc/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Mutagénesis Sitio-Dirigida , Treonina/metabolismo , Transaldolasa/genética
8.
Molecules ; 25(5)2020 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-32131464

RESUMEN

Four compounds (1-4) were isolated from the extracts of Streptomyces sp. CT37 using bioassay in conjunction with mass spectrometric molecular networking (MN) driven isolation. Their complete structures were established by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), and 1D and 2D nuclear magnetic resonance (NMR) data. Legonimide 1 was identified as a new alkaloid containing a rare linear imide motif in its structure, while compounds 2-4 were already known and their structures were elucidated as 1H-indole-3-carbaldehyde, actinopolymorphol B, (2R,3R)-1-phenylbutane-2,3-diol, respectively. The biosynthetic pathways of 1-4 were proposed based on the reported biogenesis of indole alkaloids in literature. Bioactivity tests for 1 and 2 revealed moderate growth inhibition activity against Candida albicans ATCC 10231 with MIC95 values of 21.54 µg/mL and 11.47 µg/mL, respectively.


Asunto(s)
Antifúngicos , Candida albicans/crecimiento & desarrollo , Alcaloides Indólicos , Streptomyces/química , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Alcaloides Indólicos/química , Alcaloides Indólicos/aislamiento & purificación , Alcaloides Indólicos/farmacología , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de Electrospray
9.
Molecules ; 25(2)2020 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-31936318

RESUMEN

Whole-genome sequence data of the genus Streptomyces have shown a far greater chemical diversity of metabolites than what have been discovered under typical laboratory fermentation conditions. In our previous natural product discovery efforts on Streptomyces sp. MA37, a bacterium isolated from the rhizosphere soil sample in Legon, Ghana, we discovered a handful of specialised metabolites from this talented strain. However, analysis of the draft genome of MA37 suggested that most of the encoded biosynthetic gene clusters (BGCs) remained cryptic or silent, and only a small fraction of BGCs for the production of specialised metabolites were expressed when cultured in our laboratory conditions. In order to induce the expression of the seemingly silent BGCs, we have carried out a co-culture experiment by growing the MA37 strain with the Gram-negative bacterium Pseudomonas sp. in a co-culture chamber that allows co-fermentation of two microorganisms with no direct contact but allows exchange of nutrients, metabolites, and other chemical cues. This co-culture approach led to the upregulation of several metabolites that were not previously observed in the monocultures of each strain. Moreover, the co-culture induced the expression of the cryptic indole alkaloid BGC in MA37 and led to the characterization of the known indolocarbazole alkaloid, BE-13793C 1. Neither bacterium produced compound 1 when cultured alone. The structure of 1 was elucidated by Nuclear Magnetic Resonance (NMR), mass spectrometry analyses and comparison of experimental with literature data. A putative biosynthetic pathway of 1 was proposed. Furthermore, BE-13793C 1 showed strong anti-proliferative activity against HT-29 (ATCC HTB-38) cells but no toxic effect to normal lung (ATCC CCL-171) cells. To the best of our knowledge, this is the first report for the activity of 1 against HT-29. No significant antimicrobial and anti-trypanosomal activities for 1 were observed. This research provides a solid foundation for the fact that a co-culture approach paves the way for increasing the chemical diversity of strain MA37. Further characterization of other upregulated metabolites in this strain is currently ongoing in our laboratory.


Asunto(s)
Vías Biosintéticas , Técnicas de Cocultivo/métodos , Alcaloides Indólicos/metabolismo , Metaboloma , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Bioensayo , Vías Biosintéticas/efectos de los fármacos , Vías Biosintéticas/genética , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Células HT29 , Humanos , Alcaloides Indólicos/farmacología , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Espectroscopía de Protones por Resonancia Magnética , Pseudomonas/efectos de los fármacos , Pseudomonas/metabolismo , Trypanosoma/efectos de los fármacos
10.
Molecules ; 25(3)2020 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-31979050

RESUMEN

Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 µM) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08µM) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7-12 and six siderophores 13-18.


Asunto(s)
Streptomyces/metabolismo , Antimaláricos/efectos adversos , Antineoplásicos/efectos adversos , Línea Celular Tumoral , Humanos , Familia de Multigenes/genética , Péptidos/efectos adversos , Péptidos/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray
11.
J Org Chem ; 84(24): 16323-16328, 2019 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-31729221

RESUMEN

Tricyclic carbazole is an important scaffold in many naturally occurring metabolites, as well as valuable building blocks. Here we report the reconstitution of the ring A formation of the bacterial neocarazostatin A carbazole metabolite. We provide evidence of the involvement of two unusual aromatic polyketide proteins. This finding suggests how new enzymatic activities can be recruited to specific pathways to expand biosynthetic capacities. Finally, we leveraged our bioinformatics survey to identify the untapped capacity of carbazole biosynthesis.


Asunto(s)
Carbazoles/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/química , Transferasas/metabolismo , Carbazoles/química , Carbazoles/aislamiento & purificación , Biología Computacional , Estructura Molecular
12.
Molecules ; 24(18)2019 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-31533358

RESUMEN

Drug-like molecules are known to contain many different building blocks with great potential as pharmacophores for drug discovery. The continued search for unique scaffolds in our laboratory led to the isolation of a novel Ghanaian soil bacterium, Streptomyces sp. MA37. This strain produces many bioactive molecules, most of which belong to carbazoles, pyrrolizidines, and fluorinated metabolites. Further probing of the metabolites of MA37 has led to the discovery of a new naphthacene-type aromatic natural product, which we have named accramycin A 1. This molecule was isolated using an HPLC-photodiode array (PDA) guided isolation process and MS/MS molecular networking. The structure of 1 was characterized by detailed analysis of LC-MS, UV, 1D, and 2D NMR data. Preliminary studies on the antibacterial properties of 1 using Group B Streptococcus (GBS) produced a minimum inhibitory concentration (MIC) of 27 µg/mL. This represents the first report of such bioactivity amongst the naphthacene-type aromatic polyketides, and also suggests the possibility for the further development of potent molecules against GBS based on the accramycin scaffold. A putative acc biosynthetic pathway for accramycin, featuring a tridecaketide-specific type II polyketide synthase, was proposed.


Asunto(s)
Policétidos/química , Policétidos/aislamiento & purificación , Microbiología del Suelo , Streptomyces/química , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Vías Biosintéticas , Genes Bacterianos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Familia de Multigenes , Policétidos/metabolismo , Policétidos/farmacología , Streptomyces/genética , Streptomyces/metabolismo
13.
Mar Drugs ; 17(1)2018 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-30586918

RESUMEN

A new alkaloid paenidigyamycin A (1) was obtained from the novel Ghanaian Paenibacillus sp. isolated from the mangrove rhizosphere soils of the Pterocarpus santalinoides tree growing in the wetlands of the Digya National Park, Ghana. Compound 1 was isolated on HPLC at tR = 37.0 min and its structure determined by MS, 1D, and 2D-NMR data. When tested against L. major, 1 (IC50 0.75 µM) was just as effective as amphotericin B (IC50 0.31 µM). Against L. donovani, 1 (IC50 7.02 µM) was twenty-two times less active than amphotericin B (IC50 0.32 µM), reinforcing the unique effectiveness of 1 against L. major. For T. brucei brucei, 1 (IC50 0.78 µM) was ten times more active than the laboratory standard Coptis japonica (IC50 8.20 µM). The IC50 of 9.08 µM for 1 against P. falciparum 3d7 compared to artesunate (IC50 36 nM) was not strong, but this result suggests the possibility of using the paenidigyamycin scaffold for the development of potent antimalarial drugs. Against cercariae, 1 showed high anticercaricidal activity compared to artesunate. The minimal lethal concentration (MLC) and minimal effective concentration (MEC) of the compound were 25 and 6.25 µM, respectively, while artesunate was needed in higher quantities to produce such results. However, 1 (IC50 > 100 µM) was not active against T. mobilensis.


Asunto(s)
Alcaloides/farmacología , Antiparasitarios/farmacología , Paenibacillus/química , Pterocarpus/microbiología , Alcaloides/química , Alcaloides/aislamiento & purificación , Alcaloides/uso terapéutico , Anfotericina B/farmacología , Animales , Antiparasitarios/química , Antiparasitarios/aislamiento & purificación , Antiparasitarios/uso terapéutico , Artesunato/farmacología , Cercarias/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Ghana , Imidazoles/química , Concentración 50 Inhibidora , Leishmania donovani/efectos de los fármacos , Enfermedades Parasitarias/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Rizosfera , Microbiología del Suelo , Trypanosoma brucei brucei/efectos de los fármacos , Humedales
14.
Indian J Microbiol ; 58(2): 214-221, 2018 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29651181

RESUMEN

Through the use of genomes that have undergone millions of years of evolution, marine Actinobacteria are known to have adapted to rapidly changing environmental pressures. The result is a huge chemical and biological diversity among marine Actinobacteria. It is gradually becoming a known fact that, marine Actinobacteria have the capability to produce nanoparticles which have reasonable sizes and structures with possible applications in biotechnology and pharmacology. Mycobacterium sp. BRS2A-AR2 was isolated from the aerial roots of the mangrove plant Rhizophora racemosa. The Mycobacterium was demonstrated for the first time ever to produce AuNPs with sizes that range between 5 and 55 nm. The highest level absorbance of the biosynthesized AuNPs was typical for actinobacterial strains (2.881 at 545 nm). The polydispersity index was measured as 0.207 in DLS and the zeta potential was negatively charged (- 28.3 mV). Significant vibration stretches were seen at 3314, 2358, 1635 and 667 cm-1 in FT-IR spectra. This demonstrated the possible use of small aliphatic compounds containing -COOH, -OH, -Cl and -NH2 functional groups in the stabilization of the AuNPs. The effect of the biosynthesized AuNPs on HUVEC and HeLA cell lines was measured at 48 h. IC50 values were determined at 3500 µg/ml concentration for HUVEC and HeLA cell lines at 45.25 and 53.41% respectively.

15.
Org Biomol Chem ; 15(18): 3843-3848, 2017 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-28406521

RESUMEN

Neocarazostatin A (1) is a potent free radical scavenger possessing an intriguing tricyclic carbazole nucleus with a C4 alkyl side chain attached to ring "A". Although the biosynthetic gene cluster of 1 (nzs) has been identified, and several key steps of the pathway have been well characterized, the enzyme(s) involved in the biosynthesis of the C4 unit still remains obscure. In this work, we demonstrate that three enzymes, including one (MA37-FabG) from primary fatty acid metabolism and two pathway-specific ones (NzsE and NzsF), are responsible for the formation of the side chain precursor. We show that NzsE is a free-standing acyl carrier protein (ACP), and NzsF, which is a homolog of ß-ketoacyl-acyl carrier protein synthase III (KAS III, also called FabH), catalyzes a decarboxylative condensation between an acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE. We also show that NzsF can only accept NzsE as its cognate ACP substrate, suggesting that NzsE and NzsF constitute pathway-specific KAS III enzyme pairs for the assembly line of 1. Furthermore, we have identified two FabG (the NADPH-dependent reductase) homologs from the fatty acid biosynthesis pathway that can reduce the 3-keto group of acetoacetyl-NzsE to generate a 3-hydroxybutyl-NzsE product, which is the putative intermediate for the following incorporation into 1. Therefore, our work successfully reconstitutes the biosynthetic pathway of the C4 alkyl side chain of 1in vitro, and sheds light on the potential of engineering NzsE/F for producing novel neocarazostatin analogues in the host strain.


Asunto(s)
Carbazoles/química , Carbazoles/metabolismo , Alquilación
16.
J Nat Prod ; 80(5): 1370-1377, 2017 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-28445069

RESUMEN

A new strategy for the identification of known compounds in Streptomyces extracts that can be applied in the discovery of natural products is presented. The strategy incorporates screening a database of 5555 natural products including 5098 structures from Streptomyces sp., using a high-throughput LCMS data processing algorithm that utilizes HRMS data and predicted LC retention times (tR) as filters for rapid identification of known compounds in the natural product extract. The database, named StrepDB, contains for each compound the structure, molecular formula, molecular mass, and predicted LC retention time. All identified compounds are annotated and color coded for easier visualization. It is an indirect approach to quickly assess masses (which are not annotated) that may potentially lead to the discovery of new or novel structures. In addition, a spectral database named MbcDB was generated using the ACD/Spectrus DB Platform. MbcDB contains 665 natural products, each with structure, experimental HRESIMS, MS/MS, UV, and NMR spectra. StrepDB was used to screen a mutant Streptomyces albus extract, which led to the identification and isolation of two new compounds, legonmaleimides A and B, the structures of which were elucidated with the aid of MbcDB and spectroscopic techniques. The structures were confirmed by computer-assisted structure elucidation (CASE) methods using ACD/Structure Elucidator Suite. The developed methodology suggests a pipeline approach to the dereplication of extracts and discovery of novel natural products.


Asunto(s)
Productos Biológicos/química , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas en Tándem/métodos , Bases de Datos Factuales , Estructura Molecular , Peso Molecular
17.
Org Biomol Chem ; 14(37): 8679-8684, 2016 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-27714211

RESUMEN

Although the biosynthetic pathway of Neocarazostatin A (1) has been identified, the detailed enzymatic reactions underlying the assembly of the carbazole ring still remain largely unknown. We demonstrate here that NzsH, a putative thiamine diphosphate dependent enzyme, can catalyze an acyloin coupling reaction between indole-3-pyruvate and pyruvate to generate a ß-ketoacid intermediate. Our findings thus shed light on further characterization of the unusual biosynthetic pathway of the bacterial tricyclic carbazole alkaloids.


Asunto(s)
Carbazoles/metabolismo , Streptomyces/enzimología , Tiamina Pirofosfato/metabolismo , Vías Biosintéticas , Genes Bacterianos , Indoles/metabolismo , Cinética , Familia de Multigenes , Filogenia , Ácido Pirúvico/metabolismo , Streptomyces/genética , Streptomyces/metabolismo
18.
Org Biomol Chem ; 13(37): 9585-92, 2015 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-26256511

RESUMEN

Linaridins are rare linear ribosomally-synthesized and post-translationally modified peptides (RiPPs) and only two, cypemycin and SGR-1832, in this family have been identified so far. Legonaridin 1 has been discovered as a new member of linaridins through chemical isolation, peptidogenomics, comprehensive 1- and 2-D NMR and advanced Marfey's analyses from the soil bacterium Streptomyces sp. CT34, an isolate collected from Legon, Ghana. Bioinformatics analysis of the gene cluster suggested that the biosynthesis of legonaridin 1 is different from those of cypemycin and SGR-1832. Consistent with bioinformatics and peptidogenomics analyses, 1 has a total of nine post-modifications, 8 dehydrobutyrine residues and a N,N-dimethylated N-terminus with a carboxylic acid at the C-terminus. Legonaridin 1 is structurally different from the two known linaridins comprising a new subfamily. This is the first time that NMR spectroscopy is used to establish the 2-D structure of a linaridin RiPP.


Asunto(s)
Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Ribosomas/metabolismo , Streptomyces , Secuencia de Aminoácidos , Biología Computacional , Minería de Datos , Datos de Secuencia Molecular , Familia de Multigenes , Streptomyces/genética , Streptomyces/aislamiento & purificación
19.
Angew Chem Int Ed Engl ; 54(43): 12697-701, 2015 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-26206556

RESUMEN

Pyrrolizidine alkaloids (PAs) are a group of natural products with important biological activities. The discovery and characterization of the multifunctional FAD-dependent enzyme LgnC is now described. The enzyme is shown to convert indolizidine intermediates into pyrrolizidines through an unusual ring expansion/contraction mechanism, and catalyze the biosynthesis of new bacterial PAs, the so-called legonmycins. By genome-driven analysis, heterologous expression, and gene inactivation, the legonmycins were also shown to originate from non-ribosomal peptide synthetases (NRPSs). The biosynthetic origin of bacterial PAs has thus been disclosed for the first time.


Asunto(s)
Productos Biológicos/metabolismo , Carbamatos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Alcaloides de Pirrolicidina/metabolismo , Streptomyces/enzimología , Productos Biológicos/química , Carbamatos/química , Genes Bacterianos , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Familia de Multigenes , Alcaloides de Pirrolicidina/química , Microbiología del Suelo , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo
20.
Chembiochem ; 15(3): 364-8, 2014 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-24449539

RESUMEN

The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.


Asunto(s)
Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Micromonosporaceae/genética , Nocardia/genética , Oxidorreductasas/metabolismo , Streptomyces/genética , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/metabolismo , Fluoruración , Fluoruros/química , Fluoruros/metabolismo , Cinética , Micromonosporaceae/enzimología , Familia de Multigenes , Nocardia/enzimología , Oxidorreductasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Streptomyces/enzimología
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