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1.
J Am Chem Soc ; 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38842938

ABSTRACT

Cinnamoyl-containing nonribosomal peptides (CCNPs) constitute a unique family of natural products. The enzyme mechanism for the biaryl phenol coupling reaction of the bicyclic CCNPs remains unclear. Herein, we report the discovery of two new arabinofuranosylated bicyclic CCNPs cihanmycins (CHMs) A (1) and B (2) from Amycolatopsis cihanbeyliensis DSM 45679 and the identification of the CHM biosynthetic gene cluster (cih BGC) by heterologous expression in Streptomyces lividans SBT18 to afford CHMs C (3) and D (4). The structure of 1 was confirmed by X-ray diffraction analysis. Three cytochrome P450 enzyme (CYP)-encoding genes cih26, cih32, and cih33 were individually inactivated in the heterologous host to produce CHMs E (5), F (6), and G (7), respectively. The structures of 5 and 6 indicated that Cih26 was responsible for the hydroxylation and epoxidation of the cinnamoyl moiety, and Cih32 should catalyze the ß-hydroxylation of three amino acid residues. Cih33 and its homologues DmlH and EpcH were biochemically verified to convert CHM G (7) with a monocyclic structure to a bicyclic skeleton of CHM C (3) through an intramolecular C-O phenol coupling reaction. The substrate 7-bound crystal structure of DmlH not only established the structure of 7, which was difficult for NMR analysis for displaying anomalous splitting signals, but also provided the binding mode of macrocyclic peptides recognized by these intramolecular C-O coupling CYPs. In addition, computational studies revealed a water-mediated diradical mechanism for the C-O phenol coupling reaction. These findings have shed important mechanistic insights into the CYP-catalyzed phenol coupling reactions.

2.
J Nat Prod ; 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38862138

ABSTRACT

Mangrove derived actinomycetes are a rich reservoir of bioactive natural products and play important roles in pharmaceutical chemistry. In a screen of actinomycetes from mangrove rhizosphere sedimental environments, the isolated strain Streptomyces sp. SCSIO 40068 displayed strong antibacterial activity. Further fractionation of the extract yielded four new compounds kebanmycins A-D (1-4) and two known analogues FD-594 (5) and the aglycon (6). The structures of 1-6 were determined based on extensive spectroscopic data and single-crystal X-ray diffraction analysis. 1-3 featured a fused pyranonaphthaxanthene as an integral part of a 6/6/6/6/6/6 polycyclic motif, and showed bioactivity against a series of Gram-positive bacteria and cytotoxicity to several human tumor cells. In addition, the kebanmycins biosynthetic gene cluster (keb) was identified in Streptomyces sp. SCSIO 40068, and KebMT2 was biochemically characterized as a tailoring sugar-O-methyltransferase, leading to a proposed biosynthetic route to 1-6. This study paves the way to further investigate 1 as a potential lead compound.

3.
Chem Sci ; 15(23): 8750-8755, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38873062

ABSTRACT

Quadrane sesquiterpenes featuring a distinctive tricyclic skeleton exhibit potent antimicrobial and anticancer activities. Although extensive studies have attempted to reveal the multistep carbocation rearrangement involved in the formation of the tricyclic quadrane scaffold, the exact biosynthetic pathway and chemical logic to generate the quadrane structure remains mysterious. Here we identified a novel sesquiterpene synthase that is capable of generating ß-terrecyclene possessing the quadrane scaffold and characterized the biosynthetic pathway of a representative fungal quadrane terrecyclic acid. Further mutagenesis coupled with isotopically sensitive branching studies of this ß-terrecyclene synthase provided insight into the mechanism involved in the formation of the quadrane scaffold.

4.
J Nat Prod ; 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38687877

ABSTRACT

Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aß treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.

5.
Angew Chem Int Ed Engl ; 63(22): e202403365, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38454191

ABSTRACT

Meroterpenoids of the ochraceopones family featuring a linear tetracyclic scaffold exhibit exceptional antiviral and anti-inflammatory activities. The biosynthetic pathway and chemical logic to generate this linear tetracycle, however, remain unknown. In this study, we identified and characterized all biosynthetic enzymes to afford ochraceopones and elucidated the complete biosynthetic pathway. We demonstrated that the linear tetracyclic scaffold of ochraceopones was derived from an angular tetracyclic precursor. A multifunctional cytochrome P450 OchH was validated to catalyze the free-radical-initiated carbon-carbon bond cleavage of the angular tetracycle. Then, a new carbon-carbon bond was verified to be constructed using a new aldolase OchL, which catalyzes an intramolecular aldol reaction to form the linear tetracycle. This carbon-carbon bond fragmentation and aldol reaction cascade features an unprecedented strategy for converting a common angular tetracycle to a distinctive linear tetracyclic scaffold in meroterpenoid biosynthesis.


Subject(s)
Carbon , Cytochrome P-450 Enzyme System , Carbon/chemistry , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/chemistry , Molecular Structure , Terpenes/chemistry , Terpenes/metabolism , Aldehydes/chemistry , Aldehydes/metabolism , Biocatalysis
6.
Org Lett ; 26(8): 1677-1682, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38363662

ABSTRACT

A known polycyclic tetramate macrolactam (aburatubolactam C, 3) and three new ones (aburatubolactams D-F, 4-6, respectively) were isolated from the marine-derived Streptomyces sp. SCSIO 40070. The absolute configuration of 3 was established by X-ray analysis. A combinatorial biosynthetic approach unveiled biosynthetic enzymes dictating the formation of distinct 5/5-type ring systems (such as C7-C14 cyclization by AtlB1 in 5 and C6-C13 cyclization by AtlB2 in 6) in aburatubolactams.


Subject(s)
Streptomyces , Cyclization
7.
ChemMedChem ; 19(3): e202300619, 2024 02 01.
Article in English | MEDLINE | ID: mdl-38103004

ABSTRACT

Naturally occurring resistances diminish the effectiveness of antibiotics, and present significant challenges to human health. Human activities are usually considered as the main drivers of the dissemination of antibiotic resistance, however, the origin of the clinical antibiotic resistance can be traced to the environmental microbes, and the clinically relevant resistance determinants have already pre-existed in nature before the antibiotics come into clinic. In this concept, we present the naturally occurring and widespread resistance determinants recently discovered during the biosynthesis study of bioactive compounds. These widely prevalent resistances in environmental microbes, including antibiotic producers and non-producers, advance the understanding of the origin of resistance, and provide prediction for the clinically relevant resistance to aid in the rational design of more effective drug analogues to combat resistance.


Subject(s)
Biological Products , Humans , Biological Products/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Microbial
8.
J Am Chem Soc ; 145(50): 27886-27899, 2023 12 20.
Article in English | MEDLINE | ID: mdl-38055632

ABSTRACT

The antibacterial agents deoxynybomycin (DNM) and nybomycin (NM) have a unique tetracyclic structure featuring an angularly fused 4-oxazoline ring. Here, we report the identification of key enzymes responsible for forming the 4-oxazoline ring in Embleya hyalina NBRC 13850 by comparative bioinformatics analysis of the biosynthetic gene clusters encoding structurally similar natural products DNM, deoxynyboquinone (DNQ), and diazaquinomycins (DAQs). The N-methyltransferase DnmS plays a crucial role in catalyzing the N-dimethylation of a tricyclic precursor prenybomycin to generate NM D; subsequently, the Fe(II)/α-ketoglutarate-dependent dioxygenase (Fe/αKGD) DnmT catalyzes the formation of a 4-oxazoline ring from NM D to produce DNM; finally, a second Fe/αKGD DnmU catalyzes the C-12 hydroxylation of DNM to yield NM. Strikingly, DnmT is shown to display unexpected functions to also catalyze the decomposition of the 4-oxazoline ring and the N-demethylation, thereby converting DNM back to prenybomycin, to putatively serve as a manner to control the intracellular yield of DNM. Structure modeling, site-directed mutagenesis, and quantum mechanics calculations provide mechanistic insights into the DnmT-catalyzed reactions. This work expands our understanding of the functional diversity of Fe/αKGDs in natural product biosynthesis.


Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO , Quinolones , Catalysis , Ferrous Compounds/chemistry
9.
Angew Chem Int Ed Engl ; 62(51): e202310728, 2023 Dec 18.
Article in English | MEDLINE | ID: mdl-37917570

ABSTRACT

Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.


Subject(s)
Cytochrome P-450 Enzyme System , Lactams , Cytochrome P-450 Enzyme System/metabolism , Oxidation-Reduction , Catalysis , Substrate Specificity
10.
Org Lett ; 25(34): 6346-6351, 2023 Sep 01.
Article in English | MEDLINE | ID: mdl-37606755

ABSTRACT

Tetronate antibiotics make up a growing family of natural products with a wide variety of biological activities. Herein, we report four new tetronates kongjuemycins (KJMs, 5-8) from a coral-associated actinomycete Pseudonocardia kongjuensis SCSIO 11457, and the identification and characterization of the KJM biosynthetic gene cluster (kjm) by heterologous expression, comparative genomic analysis, isotope labeling, and gene knockout studies. The biosynthesis of KJMs is demonstrated to harness diverse precursors from primary metabolism for building secondary metabolites.

11.
J Nat Prod ; 86(8): 2046-2053, 2023 08 25.
Article in English | MEDLINE | ID: mdl-37566707

ABSTRACT

Depsidones are significant in structural diversity and broad in biological activities; however, their biosynthetic pathways have not been well understood and have attracted considerable attention. Herein, we heterologously reconstituted a depsidone encoding gene cluster from Ovatospora sp. SCSIO SY280D in Aspergillus nidulans A1145, leading to production of mollicellins, a representative family of depsidones, and discovering a bifunctional P450 monooxygenase that catalyzes both ether formation and hydroxylation in the biosynthesis of the mollicellins. The functions of a decarboxylase and an aromatic prenyltransferase are also characterized to understand the tailoring modification steps. This work provides important insights into the biosynthesis of mollicellins.


Subject(s)
Cytochrome P-450 Enzyme System , Depsides , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Lactones , Ethers , Multigene Family , Biosynthetic Pathways
12.
Biochem Biophys Res Commun ; 675: 41-45, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37451216

ABSTRACT

ω-transaminase has attracted growing attention for chiral amine synthesis, although it commonly suffers from severe by-product inhibition. ω-transaminase CrmG is critical for the biosynthesis of Caerulomycin A, a natural product that possesses broad bioactivity, including immunosuppressive and anti-cancer. Compared to L-Arg, amino donor L-Glu, L-Gln or L-Ala is more preferred by CrmG. In this study, we determined the crystal structure of CrmG in complex with amino donor L-Arg, unveiling the detailed binding mode. Specifically, L-Arg exhibits an extensive contact with aromatic residues F207 and W223 on the roof of CrmG active site via cation-π network. This interaction may render the deamination by-product of L-Arg to be an inhibitor against PMP-bound CrmG by stabilizing its flexible roof, thus reducing the reactivity of L-Arg as an amino donor for CrmG. These data provide further evidence to support our previous proposal that CrmG can overcome inhibition from those by-products that are not able to stabilize the flexible roof of active site in PMP-bound CrmG. Thus, our result can not only facilitate the biosynthesis of CRM A but also be beneficial for the rational design of ω-transaminase to bypass by-product inhibition.


Subject(s)
Arginine , Transaminases , Transaminases/metabolism , Catalytic Domain
13.
J Nat Prod ; 86(4): 986-993, 2023 04 28.
Article in English | MEDLINE | ID: mdl-37042607

ABSTRACT

Fidaxomicin (Dificid) is a commercial macrolide antibiotic for treating Clostridium difficile infection. Total synthesis of fidaxomicin and its aglycone had been achieved through different synthetic schemes. In this study, an alternative biological route to afford the unique 18-membered macrolactone aglycone of fidaxomicin was developed. The promoter refactored fidaxomicin biosynthetic gene cluster from Dactylosporangium aurantiacum was expressed in the commonly used host Streptomyces albus J1074, thereby delivering five structurally diverse fidaxomicin aglycones with the corresponding titers ranging from 4.9 to 15.0 mg L-1. In general, these results validated a biological strategy to construct and diversify fidaxomicin aglycones on the basis of promoter refactoring and heterologous expression.


Subject(s)
Anti-Bacterial Agents , Streptomyces griseus , Fidaxomicin , Macrolides/metabolism , Streptomyces griseus/genetics , Multigene Family , Aminoglycosides
14.
Angew Chem Int Ed Engl ; 62(27): e202302043, 2023 07 03.
Article in English | MEDLINE | ID: mdl-37076762

ABSTRACT

Identifying new environmental resistance determinants is significant to combat rising antibiotic resistance. Herein we report the unexpected correlation of a lobophorin (LOB) resistance-related glycosidase KijX with the host-dependent chemical diversity of LOBs, by a process of glycosylation, deglycosylation and reglycosylation. KijX homologues are widespread among bacteria, archaea and fungi, and encode the same glycohydrolytic activity on LOBs. The crystal structure of AcvX (a KijX homologue) shows a similar fold to that of the glycoside hydrolase family 113 and a special negatively charged groove to accommodate and deglycosylate LOBs. Antagonistic assays indicate kijX as a defense weapon of actinomycetes to combat LOB producers in environment, reflecting an elegant coevolution relationship. Our study provides insight into the KijX-related glycosidases as preexisting resistance determinants and represents an example of resistance genes accidentally integrated into natural product assembly.


Subject(s)
Actinobacteria , Glycoside Hydrolases , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Bacteria/metabolism , Archaea , Glycosylation , Actinobacteria/metabolism
15.
ACS Synth Biol ; 12(5): 1520-1532, 2023 05 19.
Article in English | MEDLINE | ID: mdl-37084337

ABSTRACT

Ribosome-targeting oligosaccharides, everninomicins (EVNs), are promising drug leads with a unique mode of action distinct from that of currently used antibiotics in human therapy. However, the low yields in natural microbial producers hamper an efficient preparation of EVNs for detailed structure-activity relationship analysis. Herein, we enhance the production of EVNs by duplicating the biosynthetic gene cluster (BGC) in Micromonospora sp. SCSIO 07395 and thus obtain multiple EVNs that are sufficient for bioactivity evaluation. EVNs (1-5) are shown to significantly inhibit the growth of multidrug-resistant Gram-positive staphylococcal, enterococcal, and streptococcal strains and Gram-negative pathogens Acinetobacter baumannii and Vibrio cholerae, with micromolar to nanomolar potency, which are comparable or superior to vancomycin, linezolid, and daptomycin. Furthermore, the BGC duplication strategy is proven effective in stepwisely improving titers of the bioactive EVN M (5) from the trace amount to 98.6 mg L-1. Our findings demonstrate the utility of a bioengineering approach for enhanced production and chemical diversification of the medicinally promising EVNs.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents , Humans , Anti-Bacterial Agents/pharmacology , Linezolid , Bacteria , Microbial Sensitivity Tests
16.
J Nat Prod ; 86(4): 979-985, 2023 04 28.
Article in English | MEDLINE | ID: mdl-36921263

ABSTRACT

A chemical investigation of Streptomyces sp. SCSIO 40069 resulted in the isolation of a series of aromatic polyketides with rare skeletons, including five new compounds RM18c-RM18g (1-5) and three known ones (6-8). Their structures and absolute configurations were determined by diverse methods, including HRMS and NMR spectra, chemical reaction, Snatzke's method, quantum mechanical-nuclear magnetic resonance (QM-NMR), and X-ray crystallographic analysis. Compounds 1, 2, 4b, and 8 displayed moderate or weak antibacterial activities.


Subject(s)
Polyketides , Streptomyces , Molecular Structure , Streptomyces/chemistry , Polyketides/chemistry , Anti-Bacterial Agents/chemistry , Magnetic Resonance Spectroscopy
17.
Molecules ; 28(2)2023 Jan 13.
Article in English | MEDLINE | ID: mdl-36677886

ABSTRACT

Benzoxazole alkaloids exhibit a diverse array of structures and interesting biological activities. Herein we report the identification of a benzoxazole alkaloid-encoding biosynthetic gene cluster (mich BGC) in the marine-derived actinomycete Micromonospora sp. SCSIO 07395 and the heterologous expression of this BGC in Streptomyces albus. This approach led to the discovery of five new benzoxazole alkaloids microechmycin A-E (1-5), and a previously synthesized compound 6. Their structures were elucidated by HRESIMS and 1D and 2D NMR data. Microechmycin A (1) showed moderate antibacterial activity against Micrococcus luteus SCSIO ML01 with the minimal inhibitory concentration (MIC) value of 8 µg mL-1.


Subject(s)
Alkaloids , Micromonospora , Micromonospora/genetics , Micromonospora/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Alkaloids/pharmacology , Alkaloids/chemistry , Magnetic Resonance Spectroscopy , Genomics , Molecular Structure
18.
J Nat Prod ; 86(1): 76-84, 2023 01 27.
Article in English | MEDLINE | ID: mdl-36606742

ABSTRACT

The installation of halogen atoms into aromatic and less activated polyketide substrates by halogenases is a powerful strategy to tune the bioactivity, bioavailability, and reactivity of compounds. In the biosynthetic pathway of totopotensamide A (1), the halogenase TotH was confirmed in vivo to catalyze the C-4 chlorination to form the nonproteinogenic amino acid ClMeDPG. Herein, we report the isolation, structure elucidation, and bioactivity evaluation of six new deschloro totopotensamide (TPM) congeners TPMs H2-H7 (5-10) from the totH-inactivated strain and the proposed absolute configuration of the polyketide chain in TPMs using 4 as a model compound by a combination of the JBCA and bioinformatic analysis. Compounds 5, 6, 8, and 9 displayed cytotoxicity against the A549, PANC-1, Calu3, and BXPC3 cell lines with IC50 values ranging from 2.3 to 9.7 µM.


Subject(s)
Halogenation , Catalysis
19.
J Nat Prod ; 85(12): 2865-2872, 2022 12 23.
Article in English | MEDLINE | ID: mdl-36445346

ABSTRACT

Four new efrotomycins, A1-A4 (1-4), were isolated from the salt mine-derived Amycolatopsis cihanbeyliensis DSM 45679 and structurally determined. Efrotomycins A3 (3) and A4 (4) feature a tetrahydrofuran ring configured distinctly from known elfamycins. Heterologous expression of the efrotomycin gene cluster (efr BGC) in Streptomyces lividans SBT18 led to efrotomycin B1 (5), the yield of which was improved several fold upon introduction of the transporter gene efrT, a putative self-resistance determinant outside of the efr BGC.


Subject(s)
Actinomycetales , Pyridones , Multigene Family
20.
Nat Commun ; 13(1): 5386, 2022 09 14.
Article in English | MEDLINE | ID: mdl-36104338

ABSTRACT

Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the "in" conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.


Subject(s)
Mixed Function Oxygenases , Xanthones , Catalysis , Dinitrocresols , Flavin-Adenine Dinucleotide , Flavoproteins , Isoquinolines , Mixed Function Oxygenases/chemistry , Naphthoquinones , Organic Chemicals
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