RESUMEN
Biocatalysis has emerged as a valuable and reliable tool for industrial and academic societies, particularly in fields related to bioredox reactions. The cost of cofactors, especially those needed to be replenished at stoichiometric amounts or more, is the chief economic concern for bioredox reactions. In this study, a readily accessible, inexpensive, and bench-stable Hantzsch ester is verified as the viable and efficient NAD(P)H mimic by four enzymatic redox transformations, including two non-heme diiron N-oxygenases and two flavin-dependent reductases. This finding provides the potential to significantly reduce the costs of NAD(P)H-relying bioredox reactions.
Asunto(s)
NAD , NAD/metabolismo , Oxidación-Reducción , BiocatálisisRESUMEN
It is known that bacterial group II phosphopantetheinyl transferases (PPTases) usually phosphopantetheinylate acyl carrier proteins (ACPs) involved in the secondary metabolism. For example, a bacterial group II PPTase SchPPT has been known to phosphopantetheinylate only ACPs involved in secondary metabolism, such as scn ACP0-2 and scn ACP7. In this study, we found two bacterial group II PPTases, Hppt and Sppt, could phosphopantetheinylate not only scn ACP0-2 and scn ACP7, but also sch FAS ACP, an ACP involved in primary metabolism. Swapping of the N terminus and C terminus of PPTases showed that (i) both the hybrids Hppt-Sppt and Sppt-Hppt could phosphopantetheinylate sch FAS ACP but not scn ACP0-2; (ii) both the hybrids Sppt-SchPPT and SchPPT-Sppt lost abilities to phosphopantetheinylate sch FAS ACP and scn ACP0-2. Hppt and Sppt represent group II PPTases which phosphopantetheinylate both ACPs involved in primary metabolism and ACPs involved in secondary metabolism.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Metabolismo Basal , Metabolismo Secundario , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catálisis , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Filogenia , Dominios y Motivos de Interacción de Proteínas , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Benzene ring contractions are useful yet rare reactions that offer a convenient synthetic route to various valuable chemicals. However, the traditional methods of benzene contraction rely on noble-metal catalysts under extreme conditions with poor efficiency and uncontrollable selectivity. Mild-condition contractions of the benzene ring are rarely reported. This study presents a one-step, one-pot benzene ring contraction reaction mediated by an engineered nonheme diiron N-oxygenase. Using various aniline substrates as amine sources, the enzyme causes the phloroglucinol-benzene-ring contraction to afford a series of 4-cyclopentene-1,3-dione structures. A reaction detail study reveals that the nonheme diiron N-oxygenase first oxidizes the aromatic amine to a nitroso intermediate, which then attacks the phloroglucinol anion and causes benzene ring contraction. Besides, we have identified two potent antitumor compounds from the ring-contracted products.
RESUMEN
Base excision (BE) is an important yet hard-to-control biological event. Unnatural base pairs are powerful tools to revolutionize biological studies in various areas. In this paper, we report a visible-light-induced method to construct site-specific unnatural BE and show the influence of its regulation on transcription and translation levels.
Asunto(s)
Emparejamiento Base , Luz , Mutagénesis Sitio-Dirigida , Nucleótidos , Eliminación de Secuencia , Emparejamiento Base/efectos de la radiación , Nucleótidos/química , Nucleótidos/efectos de la radiación , Mutagénesis Sitio-Dirigida/métodos , Eliminación de Secuencia/efectos de la radiaciónRESUMEN
The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.
Asunto(s)
Bacterias/metabolismo , Óxido Nítrico/metabolismo , Triazoles/metabolismo , Azaguanina/metabolismo , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos , Vías Biosintéticas/genética , Genes Bacterianos/genética , Óxido Nítrico Sintasa/metabolismo , Nitrógeno , Streptomyces/enzimología , Streptomyces/genética , Streptomyces/metabolismo , Biología SintéticaRESUMEN
A novel ligand (6) for copper-catalyzed azide-alkyne cycloaddition (CuAAC) in bioconjugation has been developed. Both in vitro and in vivo experiments indicate that 6 is more efficient and less cytotoxic than the canonical CuAAC ligands. Besides, 6 is easily accessible and can be prepared at gram scale. Our study reveals that 6 might be an ideal CuAAC ligand for bioconjugations.
RESUMEN
Azoxy bond is an important chemical bond and plays a crucial role in high energy density materials. However, the biosynthetic mechanism of azoxy bond remains enigmatic. Here we report that the azoxy bond biosynthesis of azoxymycins is an enzymatic and non-enzymatic coupling cascade reaction. In the first step, nonheme diiron N-oxygenase AzoC catalyzes the oxidization of amine to its nitroso analogue. Redox coenzyme pairs then facilitate the mutual conversion between nitroso group and hydroxylamine via the radical transient intermediates, which efficiently dimerize to azoxy bond. The deficiency of nucleophilic reactivity in AzoC is proposed to account for the enzyme's non-canonical oxidization of amine to nitroso product. Free nitrogen radicals induced by coenzyme pairs are proposed to be responsible for the efficient non-enzymatic azoxy bond formation. This mechanism study will provide molecular basis for the biosynthesis of azoxy high energy density materials and other valuable azoxy chemicals.
Asunto(s)
Compuestos Azo/química , Modelos Químicos , Compuestos Azo/metabolismo , Catálisis , Simulación por Computador , Oxidación-ReducciónRESUMEN
A cryptic gene cluster, bln, was activated by genome mining in Tolypocladium ophioglossoides. This activation led to the production of balanol and eight other metabolites. Gene disruption and metabolite profile analysis showed that the biosynthesis of balanol involved the convergence of independent PKS and NRPS pathways, and a biosynthetic pathway for balanol was proposed.
Asunto(s)
Azepinas/metabolismo , Hidroxibenzoatos/metabolismo , Hypocreales/metabolismo , Vías Biosintéticas , Hypocreales/genética , Metabolómica , Familia de MultigenesRESUMEN
Aromatic azoxy compounds recently attracted wide interest for their unique liquid crystalline properties. However, biosynthetic pathways of natural azoxy products have rarely been reported. Three novel aromatic azoxy compounds, azoxymycins A, B, and C, have been isolated and identified from Streptomyces chattanoogensis L10, and their biosynthetic pathways have been reported.
Asunto(s)
Compuestos Azo/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Streptomyces/química , Compuestos Azo/química , Productos Biológicos/química , Vías Biosintéticas , Estructura MolecularRESUMEN
UNLABELLED: Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. DATABASE: Nucleotide sequence data have been submitted to the GenBank database under accession numbers KJ000382 and KJ000383.
Asunto(s)
Proteína Transportadora de Acilo/química , Aciltransferasas/química , Coenzima A/química , Sintasas Poliquetidas/química , Tacrolimus/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Familia de Multigenes , Estructura Terciaria de Proteína , Streptomyces/metabolismo , Especificidad por SustratoRESUMEN
Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.
Asunto(s)
Proteínas Bacterianas/genética , Dipéptidos/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Evolución Biológica , Magnesio/metabolismo , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.