Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Angew Chem Int Ed Engl ; 63(13): e202316133, 2024 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-38279624

RESUMEN

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.


Asunto(s)
Indenos , Oxigenasas de Función Mixta , Oxidación-Reducción , Hidroxilación , Biocatálisis
2.
J Nat Prod ; 86(11): 2448-2456, 2023 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-37870195

RESUMEN

Through genome mining efforts, two lasso peptide biosynthetic gene clusters (BGCs) within two different species of Achromobacter, a genus that contains pathogenic organisms that can infect patients with cystic fibrosis, were discovered. Using gene-refactored BGCs in E. coli, these lasso peptides, which were named achromonodin-1 and achromonodin-2, were heterologously expressed. Achromonodin-1 is naturally encoded by certain isolates from the sputum of patients with cystic fibrosis. The NMR structure of achromonodin-1 was determined, demonstrating that it is a threaded lasso peptide with a large loop and short tail structure, reminiscent of previously characterized lasso peptides that inhibit RNA polymerase (RNAP). Achromonodin-1 inhibits RNAP in vitro and has potent, focused activity toward Achromobacter pulmonis, another isolate from the sputum of a cystic fibrosis patient. These efforts expand the repertoire of antimicrobial lasso peptides and provide insights into how Achromobacter isolates from certain ecological niches interact with each other.


Asunto(s)
Achromobacter , Fibrosis Quística , Humanos , Escherichia coli , Péptidos/química , Péptidos Antimicrobianos , ARN Polimerasas Dirigidas por ADN
3.
J Biol Chem ; 294(17): 6822-6830, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30846564

RESUMEN

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.


Asunto(s)
Antibacterianos/química , Citrobacter/química , Péptidos/química , Antibacterianos/farmacología , Citrobacter/efectos de los fármacos , Descubrimiento de Drogas , Estabilidad de Medicamentos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Familia de Multigenes , Mutagénesis , Péptidos/genética , Péptidos/farmacología , Conformación Proteica
4.
Chembiochem ; 21(9): 1335-1340, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-31765515

RESUMEN

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.


Asunto(s)
Antibacterianos/farmacología , Complejo Burkholderia cepacia/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Descubrimiento de Drogas , Proteínas Citotóxicas Formadoras de Poros/farmacología , Antibacterianos/química , Complejo Burkholderia cepacia/clasificación , Humanos , Proteínas Citotóxicas Formadoras de Poros/química
5.
J Ind Microbiol Biotechnol ; 46(9-10): 1371-1379, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31165971

RESUMEN

Over the course of roughly a decade, the lasso peptide field has been transformed. Whereas new compounds were discovered infrequently via activity-driven approaches, now, the vast majority of lasso peptide discovery is driven by genome-mining approaches. This paper starts with a historical overview of the first genome-mining approaches for lasso peptide discovery, and then covers new tools that have emerged. Several examples of novel lasso peptides that have been discovered via genome mining are presented as are examples of new enzymes found associated with lasso peptide gene clusters. Finally, this paper concludes with future directions and unsolved challenges in lasso peptide genome mining.


Asunto(s)
Genoma , Péptidos/metabolismo , Humanos , Familia de Multigenes , Péptidos/química , Péptidos/genética
6.
Nat Chem ; 14(11): 1325-1334, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35982233

RESUMEN

Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain-side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the L-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.


Asunto(s)
Ácido Isoaspártico , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa , Secuencia de Aminoácidos , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Péptidos/química , Metiltransferasas/metabolismo , Ésteres
7.
ACS Chem Biol ; 14(12): 2783-2792, 2019 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-31742991

RESUMEN

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded-ring topology. The N-terminus of the peptide forms an isopeptide bond with an aspartate or glutamate side chain to create a 7-9 amino acid (aa) macrocyclic ring through which the rest of the peptide is threaded. The result is a highly constrained three-dimensional structure. Even though they share a threaded-ring feature, characterized lasso peptides vary greatly in sequence and size, ranging from 14 to 26 aa. Using genome mining, we identified a new lasso peptide gene cluster with a predicted lasso peptide that is 33 aa long. Here we report the heterologous expression of this new peptide, pandonodin, its NMR structure, and its unusual biophysical properties. Pandonodin has a long, proteolytically resistant 18-residue tail of low sequence complexity, which limits its water solubility. Within this tail is a 6 aa disulfide-bonded macrocycle that serves as a steric lock to maintain the lasso structure. This disulfide bond is unusually stable, requiring both heat and high concentrations of reductants for cleavage. Finally, we also show that segments of the C-terminal tail of pandonodin can be replaced with arbitrary sequences, allowing for the construction of pandonodin-protein fusions.


Asunto(s)
Péptidos/química , Proteobacteria/química , Secuencia de Aminoácidos , Fenómenos Biofísicos , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Péptidos/genética , Conformación Proteica , Estabilidad Proteica
8.
Chem Commun (Camb) ; 54(11): 1339-1342, 2018 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-29350227

RESUMEN

We describe a lasso peptide, albusnodin, that is post-translationally modified with an acetyl group, the first example of a lasso peptide with this modification. Using heterologous expression, we further show that the acetyltransferase colocalized with the albusnodin gene cluster is required for the biosynthesis of this lasso peptide. This type of lasso peptide is widespread in Actinobacteria with 44 examples found in currently sequenced genomes.


Asunto(s)
Proteínas Bacterianas/química , Péptidos Cíclicos/química , Streptomyces/química , Acetilación , Acetiltransferasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Péptidos Cíclicos/genética , Péptidos Cíclicos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Streptomyces/enzimología , Streptomyces/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA