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Covering: up to the end of 2023Natural nitriles comprise a small set of secondary metabolites which however show intriguing chemical and functional diversity. Various patterns of nitrile biosynthesis can be seen in animals, plants, and microorganisms with the characteristics of both evolutionary divergence and convergence. These specialized compounds play important roles in nitrogen metabolism, chemical defense against herbivores, predators and pathogens, and inter- and/or intraspecies communications. Here we review the naturally occurring nitrile-forming pathways from a biochemical perspective and discuss the biological and ecological functions conferred by diversified nitrile biosyntheses in different organisms. Elucidation of the mechanisms and evolutionary trajectories of nitrile biosynthesis underpins better understandings of nitrile-related biology, chemistry, and ecology and will ultimately benefit the development of desirable nitrile-forming biocatalysts for practical applications.
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Nitrilos , Plantas , Nitrilos/metabolismo , Nitrilos/química , Estructura Molecular , Plantas/metabolismo , AnimalesRESUMEN
A bacterial strain designated PU5-4T was isolated from the mealworm (the larvae of Tenebrio molitor) intestines. It was identified to be Gram-stain-negative, strictly aerobic, rod-shaped, non-motile, and non-spore-forming. Strain PU5-4T was observed to grow at 10-40â°C, at pH 7.0-10.0, and in the presence of 0-3.0â% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PU5-4T should be assigned to the genus Sphingobacterium. The 16S rRNA gene sequence similarity analysis showed that strain PU5-4T was closely related to the type strains of Sphingobacterium lactis DSM 22361T (98.49â%), Sphingobacterium endophyticum NYYP31T (98.11â%), Sphingobacterium soli NCCP 698T (97.69â%) and Sphingobacterium olei HAL-9T (95.73â%). The predominant isoprenoid quinone is MK-7. The major fatty acids were identified as iso-C15â:â0, iso-C17â:â03-OH and summed feature 3 (C16â:â1 ω7c and/or C16â:â1 ω6c) and summed feature 9 (iso-C17â:â0 ω9c). The polar lipids are phosphatidylethanolamine, one unidentified phospholipid, and six unidentified lipids. The genomic DNA G+C content of strain PU5-4T is 40.24 mol%. The average nucleotide identity of strain PU5-4T exhibited respective values of 73.88, 73.37, 73.36 and 70.84â% comparing to the type strains of S. lactis DSM 22361T, S. soli NCCP 698T, S. endophyticum NYYP31T and S. olei HAL-9T, which are below the cut-off level (95-96â%) for species delineation. Based on the above results, strain PU5-4T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium temoinsis sp. nov. is proposed. The type strain is PU5-4T (=CGMCC 1.61908T=JCM 36663T).
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Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano , Ácidos Grasos , Intestinos , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , Sphingobacterium , Tenebrio , Vitamina K 2 , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , ADN Bacteriano/genética , Sphingobacterium/genética , Sphingobacterium/aislamiento & purificación , Sphingobacterium/clasificación , Animales , Intestinos/microbiología , Vitamina K 2/análogos & derivados , Vitamina K 2/análisis , Tenebrio/microbiología , Fosfatidiletanolaminas , Larva/microbiología , Fosfolípidos/análisisRESUMEN
Chemically inert hydrocarbons are the primary feedstocks used in the petrochemical industry and can be converted into more intricate and valuable chemicals. However, two major challenges impede this conversion process: selective activation of C-H bonds in hydrocarbons and systematic functionalization required to synthesize complex structures. To address these issues, we developed a multi-enzyme cascade conversion system based on internal cofactor and H2O2 recycling to achieve the one-pot deep conversion from heptane to chiral (S)-2-aminoheptanoic acid under mild conditions. First, a hydrogen-borrowing-cycle-based NADH regeneration method and H2O2in situ generation and consumption strategy were applied to realize selective C-H bond oxyfunctionalization, converting heptane into 2-hydroxyheptanoic acid. Integrating subsequent reductive amination driven by the second hydrogen-borrowing cycle, (S)-2-aminoheptanoic acid was finally accumulated at 4.57 mM with eep > 99%. Hexane, octane, 2-methylheptane, and butylbenzene were also successfully converted into the corresponding chiral amino acids with eep > 99%. Overall, the conversion system employed internal cofactor and H2O2 recycling, with O2 as the oxidant and ammonium as the amination reagent to fulfill the enzymatic conversion from chemically inert hydrocarbons into chiral amino acids under environmentally friendly conditions, which is a highly challenging transformation in traditional organic synthesis.
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Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.
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Alcaligenes , Amoníaco , Hidroxilaminas , Oxidorreductasas , Alcaligenes/enzimología , Amoníaco/metabolismo , Proteínas Bacterianas/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Hidroxilaminas/metabolismo , NAD/metabolismo , Nitrógeno/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , OxígenoRESUMEN
Friedel-Crafts acylation (FCA) is a highly beneficial approach in organic chemistry for creating the important C-C bonds that are necessary for building intricate frameworks between aromatic substrates and an acyl group. However, there are few reports about enzyme catalyzed FCA reactions. In this study, 4-acyl-5-aminoimidazole alkaloids (AAIAs), streptimidazoles A-C (1-3), and the enantiopure (+)-nocarimidazole C (4) as well as their ribosides, streptimidazolesides A-D (5-8), were identified from the fermentation broth of Streptomyces sp. OUCMDZ-944 or heterologous S. coelicolor M1154 mutant. The biosynthetic gene cluster (smz) was identified, and the biosynthetic pathway of AAIAs was elucidated for the first time. In vivo and in vitro studies proved the catalytic activity of the four essential genes smzB, -C, -E, and -F for AAIAs biosynthesis and clarified the biosynthetic process of the alkaloids. The ligase SmzE activates fatty acyl groups and connects them to the acyl carrier protein (ACP) holo-SmzF. Then, the acyl group is transferred onto the key residue Cys49 of SmzB, a new Friedel-Crafts acyltransferase (FCase). Subsequently, the FCA reaction between the acyl groups and 5-aminoimidazole ribonucleotide (AIR) occurs to generate the key intermediate AAIA-nucleotides catalyzed by SmzB. Finally, the hydrolase SmzC catalyzes the N-glycosidic bond cleavage of the intermediates to form AAIAs. Structural simulation, molecular modeling, and mutational analysis of SmzB showed that Tyr26, Cys49, and Tyr93 are the key catalytic residues in the C-C bond formation of the acyl chain of AAIAs, providing mechanistic insights into the enzymatic FCA reaction.
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Aciltransferasas , Imidazoles , Aciltransferasas/química , Proteína Transportadora de Acilo/química , CatálisisRESUMEN
Cytochromes P450 are useful biocatalysts in synthetic chemistry and important bio-bricks in synthetic biology. Almost all bacterial P450s require separate redox partners for their activity, which are often expressed in recombinant Escherichia coli using multiple plasmids. However, the application of CRISPR/Cas recombineering facilitated chromosomal integration of heterologous genes which enables more stable and tunable expression of multi-component P450 systems for whole-cell biotransformations. Herein, we compared three E. coli strains W3110, JM109, and BL21(DE3) harboring three heterologous genes encoding a P450 and two redox partners either on plasmids or after chromosomal integration in two genomic loci. Both loci proved to be reliable and comparable for the model regio- and stereoselective two-step oxidation of (S)-ketamine. Furthermore, the CRISPR/Cas-assisted integration of the T7 RNA polymerase gene enabled an easy extension of T7 expression strains. Higher titers of soluble active P450 were achieved in E. coli harboring a single chromosomal copy of the P450 gene compared to E. coli carrying a medium copy pET plasmid. In addition, improved expression of both redox partners after chromosomal integration resulted in up to 80% higher (S)-ketamine conversion and more than fourfold increase in total turnover numbers.
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Escherichia coli , Ketamina , Escherichia coli/genética , Escherichia coli/metabolismo , Ketamina/metabolismo , Plásmidos/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-ReducciónRESUMEN
Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. γ-Glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide, the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in â¼23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied because of the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate glutamylmethylamide. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles.
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Ligasas de Carbono-Nitrógeno/metabolismo , Glutamatos/metabolismo , Adenosina Trifosfato/metabolismo , Catálisis , Escherichia coli/metabolismo , Ácido Glutámico/metabolismo , Magnesio/metabolismo , Metilaminas/metabolismo , Microscopía Electrónica , Rhodovulum/metabolismoRESUMEN
BACKGROUND: Plants synthesize metabolites to adapt to a continuously changing environment. Metabolite biosynthesis often occurs in response to the tissue-specific combinatorial developmental cues that are transcriptionally regulated. Polyphyllins are the major bioactive components in Paris species that demonstrate hemostatic, anti-inflammatory and antitumor effects and have considerable market demands. However, the mechanisms underlying polyphyllin biosynthesis and regulation during plant development have not been fully elucidated. RESULTS: Tissue samples of P. polyphylla var. yunnanensis during the four dominant developmental stages were collected and investigated using high-performance liquid chromatography and RNA sequencing. Polyphyllin concentrations in the different tissues were found to be highly dynamic across developmental stages. Specifically, decreasing trends in polyphyllin concentration were observed in the aerial vegetative tissues, whereas an increasing trend was observed in the rhizomes. Consistent with the aforementioned polyphyllin concentration trends, different patterns of spatiotemporal gene expression in the vegetative tissues were found to be closely related with polyphyllin biosynthesis. Additionally, molecular dissection of the pathway components revealed 137 candidate genes involved in the upstream pathway of polyphyllin backbone biosynthesis. Furthermore, gene co-expression network analysis revealed 74 transcription factor genes and one transporter gene associated with polyphyllin biosynthesis and allocation. CONCLUSIONS: Our findings outline the framework for understanding the biosynthesis and accumulation of polyphyllins during plant development and contribute to future research in elucidating the molecular mechanism underlying polyphyllin regulation and accumulation in P. polyphylla.
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Liliaceae , Saponinas , Cromatografía Líquida de Alta Presión , Liliaceae/genética , RNA-Seq , Rizoma , Saponinas/químicaRESUMEN
BACKGROUND: Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. RESULTS: In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. CONCLUSIONS: Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.
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Productos Biológicos , Glucosiltransferasas , Glucósidos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Preparaciones Farmacéuticas , Prolil Oligopeptidasas , Uridina DifosfatoRESUMEN
Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and ß-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB' as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC' and O-methyltransferase MpaG', the Golgi apparatus-associated prenyltransferase MpaA', the endoplasmic reticulum-bound oxygenase MpaB' and P450-hydrolase fusion enzyme MpaDE', and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH'. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal ß-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.
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Ácido Micofenólico/metabolismo , Aspergillus oryzae/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Redes y Vías Metabólicas , Oxidación-Reducción , Penicillium/metabolismo , Peroxisomas/metabolismo , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C-H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
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Biotecnología , Sistema Enzimático del Citocromo P-450/genética , Xenobióticos/metabolismo , Biocatálisis , Sistema Enzimático del Citocromo P-450/química , Ingeniería Metabólica , Ingeniería de Proteínas , Esteroides/biosíntesis , Esteroides/química , Especificidad por Sustrato , Xenobióticos/químicaRESUMEN
Covering: 2015 to the end of 2020 Fungal-derived polyketides, non-ribosomal peptides, terpenoids and their hybrids contribute significantly to the chemical space of total natural products. Cytochrome P450 enzymes play essential roles in fungal natural product biosynthesis with their broad substrate scope, great catalytic versatility and high frequency of involvement. Due to the membrane-bound nature, the functional and mechanistic understandings for fungal P450s have been limited for quite a long time. However, recent technical advances, such as the efficient and precise genome editing techniques and the development of several filamentous fungal strains as heterologous P450 expression hosts, have led to remarkable achievements in fungal P450 studies. Here, we provide a comprehensive review to cover the most recent progresses from 2015 to 2020 on catalytic functions and mechanisms, research methodologies and remaining challenges in the fast-growing field of fungal natural product biosynthetic P450s.
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Sistema Enzimático del Citocromo P-450/metabolismo , Hongos/enzimología , Productos Biológicos/metabolismo , Catálisis , Policétidos/metabolismo , Terpenos/metabolismoRESUMEN
Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.
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Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Podofilotoxina/uso terapéutico , Diseño de Fármacos , HumanosRESUMEN
Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and biomanufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenol phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenol substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenol phosphate was solved and compared with its complex structure with p-ethylphenol phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenol substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibilities, and the distances of the active oxygen to H pro-S and H pro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights into CreJ regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering.IMPORTANCE The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance for both biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ but also the promising directions for future engineering efforts of this enzyme toward more useful products. It also has great extendable potential to couple this multifunctional P450 enzyme with other biocatalysts (e.g., hydroxyl-based glycosylase) to access more alkylphenol-derived high-value chemicals through environment-friendly biocatalysis and biotransformation.
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Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Fenoles/metabolismo , Oxidación-Reducción , FosforilaciónRESUMEN
Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae. Here, we identify the mutant PikCD50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikCD50N can mediate the drug-metabolizing reactions using the low-cost H2O2 as a direct electron and oxygen donor.
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Peróxido de Hidrógeno , Preparaciones Farmacéuticas , Sistema Enzimático del Citocromo P-450/genética , Humanos , MacrólidosRESUMEN
A new bacterium was successfully isolated from a mangrove sediment sample in Haikou City, Hainan Province, China. The organism is a Gram-negative, rod-shaped, non-motile and strictly aerobic bacterium, named NBU-8HK146T. Strain NBU-8HK146T was able to grow at temperatures of 10-40 °C, at salinities of 0-11% (w/v) and at pH 5.5-9.5. Veoges-Proskauer, methyl red reaction and hydrolysis of Tween 20 were negative. Catalase and oxidase activities, H2S production, hydrolysis of starch, casein, Tweens 40, 60 and 80 were positive. The major cellular fatty acids were C16:0, iso-C15:0 and summed feature 9. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and two unidentified glycolipids. According to 16S rRNA gene sequence similarities, strain NBU-8HK146T shared 98.0%, 97.9%, 97.7%, 97.6% and 97.3% similarities to the species with validated name Microbulbifer taiwanensis CC-LN1-12T, Microbulbifer rhizosphaerae Cs16bT, Microbulbifer marinus Y215T, Microbulbifer donghaiensis CN85T and Microbulbifer aggregans CCB-MM1T, respectively. Phylogenetic analyses indicated that strain NBU-8HK146T formed a distinct lineage with strains Microbulbifer taiwanensis CC-LN1-12T and Microbulbifer marinus Y215T. Both digital DNA-DNA hybridization values (19.5-22.7%) and average nucleotide identity values (73.2-78.9%) between strain NBU-8HK146T and related species of genus Microbulbifer were below the species delineation cutoffs. The DNA G+C content was 58.9 mol%. Many proteins involving in the adaption of osmotic stress in the salt environment of mangrove were predicted in genome of strain NBU-8HK146T. From phenotypic, genotypic, phylogenetic and chemotaxonomic characteristics, strain NBU-8HK146T can be regarded as a new Microbulbifer species for which the name Microbulbifer hainanensis. The type strain is NBU-8HK146T (= KCTC 82226T = MCCC 1K04737T).
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Ácidos Grasos , Sedimentos Geológicos , Adolescente , Técnicas de Tipificación Bacteriana , Niño , China , ADN Bacteriano/genética , Ácidos Grasos/análisis , Gammaproteobacteria , Humanos , Fosfolípidos , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Bafilomycin A1 is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H+-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (baf) from the marine bacterium Streptomyces lohii ATCC BAA-1276, wherein a luxR family regulatory gene orf1 and an afsR family regulatory gene bafG were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of orf1 and bafG for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type S. lohii strain, the knockout of either orf1 or bafG completely abolished the production of bafilomycins. The overexpression of orf1 or bafG led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically engineered S. lohii strain (SLO-08) with orf1 overexpression and inactivation of the biosynthetic genes orf2 and orf3, solely produced bafilomycin A1 with the titer of 535.1 ± 25.0 mg/L in an optimized fermentation medium in shaking flasks. This recombinant strain holds considerable application potential in large-scale production of bafilomycin A1 for new drug development.
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Ingeniería Genética/métodos , Macrólidos/metabolismo , Biosíntesis de Proteínas/fisiología , Streptomyces/genética , Streptomyces/metabolismo , Organismos Acuáticos/genética , Organismos Acuáticos/metabolismo , Técnicas de Inactivación de Genes/métodosRESUMEN
CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H2 O2 as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleTJE , P450SPα , P450BSß ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleTJE , and molecular docking results suggest that the unprecedented selectivity is due to OleTJE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSß regardless of the cis/trans-configuration of substrates. The combined H218 O2 tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compoundâ I-mediated aldehyde formation in which the active site water derived from H2 O2 activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.
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Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Insaturados/metabolismo , Bacillus subtilis/enzimología , Sistema Enzimático del Citocromo P-450/química , Ácidos Grasos Insaturados/química , Peróxido de Hidrógeno/metabolismo , Hidroxilación , Oxigenasas de Función Mixta/metabolismo , Simulación de Dinámica Molecular , Peroxidasas/metabolismo , Teoría Cuántica , Sphingomonas/enzimología , Staphylococcaceae/enzimología , Estereoisomerismo , Especificidad por SustratoRESUMEN
The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.
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Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Sulfurtransferasas/metabolismo , Actinoplanes/genética , Actinoplanes/metabolismo , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Indoles/análisis , Indoles/química , Indoles/metabolismo , Familia de Multigenes , Pyrococcus/enzimología , Pyrococcus/genética , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/genética , Ubiquitinación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.