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1.
Nature ; 590(7846): 463-467, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536618

RESUMO

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.


Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Furanos/química , Hormônios/química , Hormônios/classificação , Hormônios/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/classificação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relação Estrutura-Atividade
2.
Nucleic Acids Res ; 51(3): 1488-1499, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36718812

RESUMO

Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.


Assuntos
Inibidores de Proteassoma , Saccharomyces cerevisiae , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Sequência de Bases , Saccharomyces cerevisiae/genética , Família Multigênica
3.
J Am Chem Soc ; 146(9): 6114-6124, 2024 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-38389455

RESUMO

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.


Assuntos
Produtos Biológicos , Imidazóis , Macrolídeos , Polienos , Policetídeos , Sulfonamidas , Tiofenos , Policetídeo Sintases/metabolismo , Aciltransferases , Antibacterianos , Policetídeos/metabolismo , Produtos Biológicos/metabolismo
4.
Chembiochem ; 22(13): 2262-2265, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33851500

RESUMO

Aromatic nitration reactions are a cornerstone of organic chemistry, but are challenging to scale due to corrosive reagents and elevated temperatures. The cytochrome P450 TxtE nitrates the indole 4-position of l-tryptophan at room temperature using NO, O2 and NADPH, and has potential to be developed into a useful aromatic nitration biocatalyst. However, its narrow substrate scope (requiring both the α-amino acid and indole functionalities) have hindered this. Screening of an R59 mutant library of a TxtE-reductase fusion protein identified a variant (R59C) that nitrates tryptamine, which is not accepted by native TxtE. This variant exhibits a broader substrate scope than the wild type enzyme and is able to nitrate a range of tryptamine analogues, with significant alterations to the aromatic and aminoethyl moieties.


Assuntos
Nitratos/metabolismo , Triptofano/metabolismo , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/metabolismo , Estrutura Molecular , Oxirredução , Especificidade por Substrato
5.
J Am Chem Soc ; 142(37): 15764-15779, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32811149

RESUMO

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Compostos Férricos/metabolismo , Nitrocompostos/metabolismo , Ácido Peroxinitroso/metabolismo , Biocatálise , Teoria da Densidade Funcional , Compostos Férricos/química , Modelos Moleculares , Conformação Molecular , Nitrocompostos/química , Ácido Peroxinitroso/química
6.
J Am Chem Soc ; 141(1): 216-222, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30516965

RESUMO

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation, and natural product biosynthesis. Here we report biochemical, structural, and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the π-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/metabolismo , Piperazinas/metabolismo , Sítios de Ligação , Biocatálise , Hidroxilação , Indóis/química , Modelos Moleculares , Piperazinas/química , Conformação Proteica , Especificidade por Substrato
7.
Nat Prod Rep ; 35(7): 622-632, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29651484

RESUMO

Covering: up to the end of 2017 The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.


Assuntos
Produtos Biológicos/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Oxigenases/química , Oxigenases/metabolismo , Ciclização , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Heme , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Hidroxilação , Prodigiosina/análogos & derivados , Prodigiosina/biossíntese , Prodigiosina/química , Pirróis/química , Pirróis/metabolismo , Pirrolnitrina/biossíntese , Compostos de Espiro/metabolismo
8.
Nat Chem Biol ; 12(3): 194-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26807714

RESUMO

Pyridoxal 5'-phosphate (PLP)-dependent enzymes have wide catalytic versatility but are rarely known for their ability to react with oxygen to catalyze challenging reactions. Here, using in vitro reconstitution and kinetic analysis, we report that the indolmycin biosynthetic enzyme Ind4, from Streptomyces griseus ATCC 12648, is an unprecedented O2- and PLP-dependent enzyme that carries out a four-electron oxidation of L-arginine, including oxidation of an unactivated carbon-carbon (C-C) bond. We show that the conjugated product of this reaction, which is susceptible to nonenzymatic deamination, is efficiently intercepted and stereospecifically reduced by the partner enzyme Ind5 to give D-4,5-dehydroarginine. Thus, Ind4 couples the redox potential of O2 with the ability of PLP to stabilize anions to efficiently oxidize an unactivated C-C bond, with the subsequent stereochemical inversion by Ind5 preventing off-pathway reactions. Altogether, these results expand our knowledge of the catalytic versatility of PLP-dependent enzymes and enrich the toolbox for oxidative biocatalysis.


Assuntos
Carbono/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Fosfato de Piridoxal/química , Arginina/química , Catálise , Desaminação , Cinética , Conformação Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estereoisomerismo , Streptomyces griseus/enzimologia
9.
Proc Natl Acad Sci U S A ; 112(9): 2717-22, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25730866

RESUMO

The bacterial tryptophanyl-tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetate-CoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Oxazolona/análogos & derivados , Rhodococcus/metabolismo , Streptomyces griseus/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sistema Livre de Células/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Indóis/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Oxazolona/metabolismo , Rhodococcus/genética , Streptomyces griseus/genética
10.
J Am Soc Mass Spectrom ; 35(7): 1490-1496, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830009

RESUMO

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H]12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an Elab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H]12+ EF-Tu ion showed similar qualitative behavior to the experimental results.


Assuntos
Antibacterianos , Simulação de Dinâmica Molecular , Fator Tu de Elongação de Peptídeos , Desdobramento de Proteína , Espectrometria de Massas por Ionização por Electrospray , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Antibacterianos/química
11.
ACS Infect Dis ; 10(9): 3378-3391, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39189814

RESUMO

Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, particularly their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinomycetota extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 µM. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster (BGC). A cytochrome P450 encoded by this gene cluster catalyzes the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyze the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. One of these encodes a glycosyl transferase that was shown to catalyze attachment of a D-glucose residue to nosiheptide in vitro. The discovery of the persiathiacins and their BGC thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.


Assuntos
Família Multigênica , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Humanos , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Testes de Sensibilidade Microbiana , Antituberculosos/farmacologia , Antituberculosos/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Glicosilação , Actinobacteria/metabolismo , Actinobacteria/genética , Vias Biossintéticas , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-29685972

RESUMO

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in Streptomyces species involves the DesD-catalysed condensation of various N-acylated derivatives of N-hydroxycadaverine with two molecules of N-succinyl-N-hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N-acyl-N-hydroxycadaverine substrates of DesD from N-hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N-acylation of N-hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N-succinyl-N-hydroxycadaverine and one of N-acetyl-N-hydroxycadaverine) was also determined. This showed that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely function. This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.


Assuntos
Aciltransferases/metabolismo , Desferroxamina/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Hidroxâmicos/metabolismo , Streptomyces coelicolor/metabolismo , Compostos Férricos/metabolismo , Ferro , Receptores de Lipoproteínas/metabolismo , Streptomyces coelicolor/enzimologia
13.
Curr Opin Chem Biol ; 35: 73-79, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27632683

RESUMO

Class B radical S-adenosylmethionine (SAM) methylases are notable for their ability to catalyse methylation reactions in the biosynthesis of a wide variety of natural products, including polyketides, ribosomally biosynthesised and post-translationally modified peptides (RiPPs), nonribosomal peptides (NRPs), aminoglycosides, ß-lactams, phosphonates, enediynes, aminocoumarins and terpenes. Here, we discuss the diversity of substrates and catalytic mechanism utilised by such enzymes, highlighting the stereochemical course of methylation reactions at un-activated carbon centres and the ability of some members of the family to catalyse multiple methylations.


Assuntos
Produtos Biológicos/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Metilação
14.
Chem Biol ; 22(3): 317-28, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25794436

RESUMO

Tryptophan, the most chemically complex and the least abundant of the 20 common proteinogenic amino acids, is a biosynthetic precursor to a large number of complex microbial natural products. Many of these molecules are promising scaffolds for drug discovery and development. The chemical features of tryptophan, including its ability to undergo enzymatic modifications at almost every atom in its structure and its propensity to undergo spontaneous, non-enzyme catalyzed chemistry, make it a unique biological precursor for the generation of chemical complexity. Here, we review the pathways that enable incorporation of tryptophan into complex metabolites in bacteria, with a focus on recently discovered, unusual metabolic transformations.


Assuntos
Bactérias/metabolismo , Triptofano/metabolismo , Bactérias/química , Triptofano/biossíntese , Triptofano/química
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