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1.
Cell Chem Biol ; 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34474009

RESUMO

Mechercharmycin A (MCM-A) is a marine natural product belonging to a family of polyazole cyclopeptides with remarkable bioactivities and unique structures. Identification, heterologous expression, and genetic characterizations of the MCM biosynthetic gene cluster in Bacillus subtilis revealed that it is a ribosomally synthesized and post-translationally modified peptide (RiPP) possessing complex with distinctive modifications. Based on this heterologous expression system, two MCM analogs with comparable antitumor activity are generated by engineering the biosynthetic pathway. Combinatorial co-production of a precursor peptide with different modifying enzymes in Escherichia coli identifies a different timing of modifications, showing that a tRNAGlu-dependent highly regioselective dehydration is the first modification step, followed by polyazole formation through heterocyclization and dehydrogenation in an N- to C-terminal direction. Therefore, a rational biosynthetic pathway of MCMs is proposed, which unveils a subfamily of azol(in)e-containing RiPPs and sets the stage for further investigations of the enzymatic mechanism and synthetic biology.

2.
J Org Chem ; 86(16): 10943-10945, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34412479
3.
Org Lett ; 23(6): 2342-2346, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33683897

RESUMO

Rifamycins have been clinically utilized against mycobacterial infections for more than 50 years; however, their biosynthesis has not been fully elucidated. Here, on the basis of in vivo gene deletions, in vitro enzyme assays, isotope labeling, and site-directed mutations, we found that a flavin-dependent monooxygenase encoded by a rifamycin biosynthetic gene cluster, Rif-Orf17, not only converted the naphthoquinone chromophore of rifamycin S into benzo-γ-pyrone but also linearized rifamycin SV through phenolic hydroxylation. Both oxidation routes lead to inactivation of rifamycins.

4.
Front Microbiol ; 11: 971, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582048

RESUMO

Rifamycin derivatives, such as rifampicin, have potent antibiotic activity and have long been used in the clinic as mainstay components for the treatment of tuberculosis, leprosy, and AIDS-associated mycobacterial infections. However, the extensive usage of these antibiotics has resulted in the rapid development of bacterial resistance. The resistance mechanisms mainly include mutations of the rifamycin target RNA polymerase of bacteria and enzymatic modifications of rifamycin antibiotics. One modification is the recently characterized rifamycin degradation catalyzed by Rox enzymes, which belong to the widely occurring flavin monooxygenases. Intriguingly, our recent sequence analysis revealed the rifamycin producers also encode Rox homologs that are not yet characterized. In this work, we expanded the study of the Rox-catalyzed rifamycin degradation. We first showed that the Rox proteins from rifamycin producers have the enzymatic rifamycin SV-degrading activity. Then we used the structurally diverse rifamycin compounds rifampicin and 16-demethylrifamycin W to probe the substrate scope and found that they each have a slightly different substrate scope. Finally, we demonstrated that Rox proteins can also catalyze the transformation of 16-demethylsalinisporamycin to 16-demethylsaliniketal A. Since 16-demethylsalinisporamycin and 16-demethylsaliniketal A are the counterpart analogs of salinisporamycin and saliniketal A, our biochemical findings not only uncover a previously uncharacterized self-resistance mechanism in the rifamycin producers, but also bridge the gap between the biosynthesis of the potential antitumor compound saliniketal A.

5.
J Am Chem Soc ; 142(13): 5996-6000, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32167762

RESUMO

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.


Assuntos
Proteínas de Bactérias/metabolismo , Nucleosídeos/metabolismo , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Streptomyces/metabolismo , Açúcares/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Família Multigênica , Nucleosídeos/genética , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Streptomyces/genética
6.
Nat Prod Rep ; 37(1): 17-28, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31290896

RESUMO

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp3 carbon or add onto an sp2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.


Assuntos
Enzimas/química , Enzimas/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coproporfirinogênio Oxidase/química , Coproporfirinogênio Oxidase/metabolismo , Duocarmicinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Hidrogênio , Metilação , Peptídeos Cíclicos/metabolismo , Policetídeos/metabolismo , Proteínas Metiltransferases/metabolismo , Tiazóis/metabolismo
7.
Org Lett ; 22(1): 150-154, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31829601

RESUMO

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.


Assuntos
Aclarubicina/análogos & derivados , Antibióticos Antineoplásicos/farmacologia , Policetídeo Sintases/metabolismo , Aclarubicina/biossíntese , Aclarubicina/química , Aclarubicina/farmacologia , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Streptomyces/química , Streptomyces/metabolismo
8.
Angew Chem Int Ed Engl ; 58(50): 18046-18054, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31553109

RESUMO

One biosynthetic gene cluster (BGC) usually governs the biosynthesis of a series of compounds exhibiting either the same or similar molecular scaffolds. Reported here is a multiplex activation strategy to awaken a cryptic BGC associated with tetracycline polyketides, resulting in the discovery of compounds having different core structures. By constitutively expressing a positive regulator gene in tandem mode, a single BGC directed the biosynthesis of eight aromatic polyketides with two types of frameworks, two pentacyclic isomers and six glycosylated tetracyclines. The proposed biosynthetic pathway, based on systematic gene inactivation and identification of intermediates, employs two sets of tailoring enzymes with a branching point from the same intermediate. These findings not only provide new insights into the role of tailoring enzymes in the diversification of polyketides, but also highlight a reliable strategy for genome mining of natural products.


Assuntos
Família Multigênica , Policetídeos/química , Policetídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Perfilação da Expressão Gênica , Genes Bacterianos , Microrganismos Geneticamente Modificados , Estrutura Molecular , Mutação , Regiões Promotoras Genéticas
9.
Org Lett ; 21(9): 3148-3152, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30990701

RESUMO

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.


Assuntos
Antibacterianos/biossíntese , Família Multigênica , Nucleosídeos/biossíntese , Purinas/biossíntese , Açúcares/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/química , Vias Biossintéticas/genética , Cicloleucina/química , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Nucleosídeos/química , Purinas/química , Streptomyces/genética , Streptomyces/metabolismo
11.
Org Lett ; 21(7): 2322-2325, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30855966

RESUMO

The biosynthetic gene cluster of antitumor antibiotic LL-D49194α1 (LLD) was identified and comparatively analyzed with that of trioxacarcins. The tailoring genes encoding glycosyltransferase, methyltransferase and cytochrome P450 were systematically deleted, which led to the discovery of eight compounds from the mutants. Preliminary pharmaceutical evaluation revealed two intermediates exhibiting higher cytotoxicity, stability and solubility. These results highlighted the modification pathway for LLD biosynthesis, and provided highly potent, structurally simplified "unnatural" natural products with improved druggability.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Metiltransferases/metabolismo , Antibióticos Antineoplásicos/química , Produtos Biológicos/química , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/química , Metiltransferases/química , Estrutura Molecular , Família Multigênica
12.
Org Lett ; 21(5): 1374-1378, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30763106

RESUMO

The biosynthetic gene clusters for herbicidins ( hbc) and aureonuclemycin ( anm) were identified in Streptomyces sp. KIB-027 and Streptomyces aureus, respectively. The roles of genes possibly involved in post-core-assembly steps in herbicidin biosynthesis in these clusters and a related her cluster were studied. Through systematic gene deletions, structural elucidation of the accumulated intermediates in the mutants, and in vitro verification of the encoded enzymes, the peripheral modification pathway for herbicidin biosynthesis is now fully established.


Assuntos
Antibacterianos/química , Nucleosídeos de Purina/biossíntese , Streptomyces/química , Estrutura Molecular
13.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530711

RESUMO

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.


Assuntos
Aminoglicosídeos/biossíntese , Aminoglicosídeos/genética , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Descoberta de Drogas , Micromonospora/genética , Micromonospora/metabolismo , Aminoglicosídeos/farmacologia , Sequência de Bases , Vias Biossintéticas/genética , Deleção de Genes , Lactamas Macrocíclicas/metabolismo , Família Multigênica/genética , Policetídeos/metabolismo , Rifamicinas/biossíntese , Metabolismo Secundário/genética
15.
Proc Natl Acad Sci U S A ; 115(44): 11232-11237, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30327344

RESUMO

Understanding how antibiotic-producing bacteria deal with highly reactive chemicals will ultimately guide therapeutic strategies to combat the increasing clinical resistance crisis. Here, we uncovered a distinctive self-defense strategy featured by a secreted oxidoreductase NapU to perform extracellularly oxidative activation and conditionally overoxidative inactivation of a matured prodrug in naphthyridinomycin (NDM) biosynthesis from Streptomyces lusitanus NRRL 8034. It was suggested that formation of NDM first involves a nonribosomal peptide synthetase assembly line to generate a prodrug. After exclusion and prodrug maturation, we identified a pharmacophore-inactivated intermediate, which required reactivation by NapU via oxidative C-H bond functionalization extracellularly to afford NDM. Beyond that, NapU could further oxidatively inactivate the NDM pharmacophore to avoid self-cytotoxicity if they coexist longer than necessary. This discovery represents an amalgamation of sophisticatedly temporal and spatial shielding mode conferring self-resistance in antibiotic biosynthesis from Gram-positive bacteria.


Assuntos
Antibacterianos/metabolismo , Pró-Fármacos/metabolismo , Streptomyces/metabolismo , Naftiridinas/metabolismo , Oxirredução , Oxirredutases/metabolismo , Peptídeo Sintases/metabolismo
16.
Angew Chem Int Ed Engl ; 57(41): 13475-13479, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30151879

RESUMO

Aromatic-fused γ-pyrones are structural features of many bioactive natural products and valid scaffolds for medicinal chemistry. However, the enzymology of their formation has not been completely established. Now it is demonstrated that TxnO9, a CalC-like protein belonging to a START family, functions as an unexpected anthraquinone-γ-pyrone synthase involved in the biosynthesis of antitumor antibiotic trioxacarcin A (TXN-A). Structural analysis by NMR identified a likely substrate/product-binding mode and putative key active sites of TxnO9, which allowed an enzymatic mechanism to be proposed. Moreover, a subset of uncharacterized homologous proteins bearing an unexamined Lys-Thr dyad exhibit the same function. Therefore, the functional assignment and mechanistic investigation of this γ-pyrone synthase elucidated an undescribed step in TXN-A biosynthesis, and the discovery of this new branch of polyketide heterocyclases expands the functions of the START superfamily.


Assuntos
Aminoglicosídeos/biossíntese , Antraquinonas/química , Antibióticos Antineoplásicos/biossíntese , Ligases/metabolismo , Policetídeos/metabolismo , Pironas/química , Aminoglicosídeos/química , Antibióticos Antineoplásicos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular
17.
Nat Commun ; 9(1): 2771, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018376

RESUMO

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.


Assuntos
Antibióticos Antineoplásicos/biossíntese , Proteínas de Bactérias/metabolismo , Ciclopropanos/metabolismo , Indóis/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Clonagem Molecular , Duocarmicinas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Metiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , Streptomyces/genética , Enxofre/metabolismo
18.
Chembiochem ; 19(19): 2002-2022, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30039582

RESUMO

Oxygen-containing heterocycles are widely encountered in natural products that display diverse pharmacological properties and have potential benefits to human health. The formation of O-heterocycles catalyzed by different types of enzymes in the biosynthesis of natural products not only contributes to the structural diversity of these compounds, but also enriches our understanding of nature's ability to construct complex molecules. This minireview focuses on the various modes of enzymatic O-heterocyclization identified in natural product biosynthesis and summarizes the possible mechanisms involved in ring closure.


Assuntos
Produtos Biológicos/metabolismo , Enzimas , Compostos Heterocíclicos/metabolismo , Oxigênio/metabolismo , Catálise , Ciclização , Enzimas/química , Enzimas/metabolismo
19.
Nat Commun ; 9(1): 2342, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29904078

RESUMO

Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C-O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.


Assuntos
Rifamicinas/biossíntese , Clonagem Molecular , DNA/química , Fermentação , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mutação , NADP/química , Especificidade por Substrato
20.
Angew Chem Int Ed Engl ; 57(3): 719-723, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29194897

RESUMO

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe2+ -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.


Assuntos
Antibacterianos/metabolismo , Oxirredutases/química , Antibacterianos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Carbono/química , Cromatografia Líquida , Dimerização , Genes Bacterianos , Hidrogênio/química , Espectrometria de Massas , Família Multigênica , Peptídeo Sintases/metabolismo , RNA de Transferência/química , Streptomyces/genética
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