RESUMO
Iron and 2-oxoglutarate dependent (Fe/2OG) enzymes exhibit an exceedingly broad reaction repertoire. The most prevalent reactivity is hydroxylation, but many other reactivities have also been discovered in recent years, including halogenation, desaturation, epoxidation, endoperoxidation, epimerization, and cyclization. To fully explore the reaction mechanisms that support such a diverse reactivities in Fe/2OG enzyme, it is necessary to utilize a multi-faceted research methodology, consisting of molecular probe design and synthesis, in vitro enzyme assay development, enzyme kinetics, spectroscopy, protein crystallography, and theoretical calculations. By using such a multi-faceted research approach, we have explored reaction mechanisms of desaturation and epoxidation catalyzed by a bi-functional Fe/2OG enzyme, AsqJ. Herein, we describe the experimental protocols and computational workflows used in our studies.
Assuntos
Ferro , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ferro/química , Ferro/metabolismo , Cinética , Cristalografia por Raios X/métodos , Ensaios Enzimáticos/métodos , Hidroxilação , Modelos MolecularesRESUMO
A wax-patterned paper analytical device (µPAD) has been developed for point-of-care colourimetric testing of serum glutamic oxaloacetic transaminase (SGOT). The detection method was based on the transamination reaction of aspartate with α-ketoglutarate, leading to the formation of oxaloacetate which reacts with the reagent Fast Blue BB salt and forms a cavern pink colour. The intensity of the cavern pink colour grows as the concentration of SGOT increases. UV-visible spectroscopy was utilized to optimize reaction conditions, and the optimized reagents were dropped onto the wax-patterned paper. The coloured PADs, after the addition of SGOT, have been photographed, and a colour band has been generated to correlate the SGOT concentration visually. The images were used to calculate the intensity values using ImageJ software, which inturn was used to calculate the SGOT concentration. The PADs were also tested with serum samples, and SGOT spiked serum samples. The PAD could detect the SGOT concentration ranging from 5 to 200 U/L. The analysis yielded highly accurate results with less than 6% relative error compared to the clinical sample. This colourimetric test demonstrated exceptional selectivity in the presence of other biomolecules in the blood serum, with a detection limit of 2.77 U/L and a limit of quantification of 9.25 U/L. Additionally, a plasma separation membrane was integrated with the PAD to directly test SGOT from finger-prick blood samples.
Assuntos
Aspartato Aminotransferases , Colorimetria , Testes Imediatos , Humanos , Aspartato Aminotransferases/sangue , Colorimetria/métodos , Papel , Limite de Detecção , Ácidos Cetoglutáricos/sangue , Ácidos Cetoglutáricos/química , Ácido Aspártico/sangue , Ácido Aspártico/químicaRESUMO
Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.
Assuntos
Aziridinas , Ciclopropanos , Ácidos Cetoglutáricos , Ciclopropanos/química , Ciclopropanos/metabolismo , Aziridinas/química , Aziridinas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/química , Ferro/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Biocatálise , Ensaios Enzimáticos/métodos , CatáliseRESUMO
Histone lysine demethylase (KDM), AlkB homolog (ALKBH), and Ten-Eleven Translocation (TET) proteins are members of the 2-Oxoglutarate (2OG) and ferrous iron-dependent oxygenases, each of which harbors a catalytic domain centered on a double-stranded ß-helix whose topology restricts the regions directly involved in substrate binding. However, they have different catalytic functions, and the deeply structural biological reasons are not yet clear. In this review, the catalytic domain features of the three protein families are summarized from both sequence and structural perspectives. The construction of the phylogenetic tree and comparison of the structure show ten relatively conserved ß-sheets and three key regions with substantial structural differences. We summarize the relationship between three key regions of remarkable differences and the substrate compatibility of the three protein families. This review facilitates research into substrate-selective inhibition and bioengineering by providing new insights into the catalytic domains of KDM, ALKBH, and TET proteins.
Assuntos
Domínio Catalítico , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Humanos , Modelos Moleculares , Filogenia , Especificidade por Substrato , Ferro/química , Ferro/metabolismo , Animais , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Sequência de AminoácidosRESUMO
In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ1-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO2. We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding ß-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives.
Assuntos
Proteínas de Bactérias , Etilenos , Ácidos Cetoglutáricos , Pseudomonas , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Etilenos/metabolismo , Etilenos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Liases/metabolismo , Liases/química , Liases/genética , Arginina/metabolismo , Arginina/química , OxirreduçãoRESUMO
Aspartyl/asparaginyl hydroxylase (AspH) catalyzes the post-translational hydroxylations of vital human proteins, playing an essential role in maintaining their biological functions. Single-point mutations in the Second Coordination Sphere (SCS) and long-range (LR) residues of AspH have been linked to pathological conditions such as the ophthalmologic condition Traboulsi syndrome and chronic kidney disease (CKD). Although the clinical impacts of these mutations are established, there is a critical knowledge gap regarding their specific atomistic effects on the catalytic mechanism of AspH. In this study, we report integrated computational investigations on the potential mechanistic implications of four mutant forms of human AspH with clinical importance: R735W, R735Q, R688Q, and G434V. All the mutant forms exhibited altered binding interactions with the co-substrate 2-oxoglutarate (2OG) and the main substrate in the ferric-superoxo and ferryl complexes, which are critical for catalysis, compared to the wild-type (WT). Importantly, the mutations strongly influence the energetics of the frontier molecular orbitals (FMOs) and, thereby, the activation energies for the hydrogen atom transfer (HAT) step compared to the WT AspH. Insights from our study can contribute to enzyme engineering and the development of selective modulators for WT and mutants of AspH, ultimately aiding in treating cancers, Traboulsi syndrome and, CKD.
Assuntos
Ácidos Cetoglutáricos , Oxigenases de Função Mista , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Mutação , Biocatálise , Compostos Ferrosos/química , Compostos Ferrosos/metabolismoRESUMO
The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Ferro , Ácidos Cetoglutáricos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/metabolismo , Ferro/química , Humanos , Especificidade por Substrato , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/química , Conformação Proteica , Uracila/metabolismo , Uracila/análogos & derivados , Uracila/química , Simulação de Dinâmica Molecular , Timina/análogos & derivadosRESUMO
Severe bleeding from deep and irregular wounds poses a significant challenge in prehospital and surgical settings. To address this issue, we developed a novel chitosan-based hemostatic dressing with a magnetic targeting mechanism using Fe3O4, termed bovine serum albumin-modified Fe3O4 embedded in porous α-ketoglutaric acid/chitosan (BSA/Fe3O4@KA/CS). This dressing enhances hemostasis by magnetically guiding the agent to the wound site. In vitro, the hemostatic efficacy of BSA/Fe3O4@KA/CS is comparable to that of commercial chitosan (Celox™) and is not diminished by the modification. In vivo, BSA/Fe3O4@KA/CS demonstrated superior hemostatic performance and reduced blood loss compared to Celox™. The hemostatic mechanism of BSA/Fe3O4@KA/CS includes the concentration of solid blood components through water absorption, adherence to blood cells, and activation of the endogenous coagulation pathway. Magnetic field targeting is crucial in directing the dressing to deep hemorrhagic sites. Additionally, safety assessments have confirmed the biocompatibility and biodegradability of BSA/Fe3O4@KA/CS. In conclusion, we introduce a novel approach to modify chitosan using magnetic guidance for effective hemostasis, positioning BSA/Fe3O4@KA/CS as a promising candidate for managing various wounds.
Assuntos
Bandagens , Quitosana , Hemostáticos , Soroalbumina Bovina , Quitosana/química , Soroalbumina Bovina/química , Animais , Hemostáticos/química , Hemostáticos/farmacologia , Porosidade , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/farmacologia , Bovinos , Masculino , Hemorragia/tratamento farmacológico , Hemorragia/terapia , CamundongosRESUMO
N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 â¼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.
Assuntos
Ferro , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/metabolismo , Ferro/química , Hidroxilação , Ciclização , Oxigenases/metabolismo , Oxigenases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
Nonheme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases 2A (KDM2A) catalyze the demethylation of the mono- or dimethylated lysine 36 residue in the histone H3 peptide (H3K36me1/me2), which plays a crucial role in epigenetic regulation and can be involved in many cancers. Although the overall catalytic mechanism of KDMs has been studied, how KDM2 catalysis takes place in contrast to other KDMs remains unknown. Understanding such differences is vital for enzyme redesign and can help in enzyme-selective drug design. Herein, we employed molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) to explore the complete catalytic mechanism of KDM2A, including dioxygen diffusion and binding, dioxygen activation, and substrate oxidation. Our study demonstrates that the catalysis of KDM2A is controlled by the conformational change of the second coordination sphere (SCS), specifically by a change in the orientation of Y222, which unlocks the 2OG rearrangement from off-line to in-line mode. The study demonstrates that the variant Y222A makes the 2OG rearrangement more favorable. Furthermore, the study reveals that it is the size of H3K36me3 that prevents the 2OG rearrangement, thus rendering the enzyme inactivity with trimethylated lysine. Calculations show that the SCS and long-range interacting residues that stabilize the HAT transition state in KDM2A differ from those in KDM4A, KDM7B, and KDM6A, thus providing the basics for the enzyme-selective redesign and modulation of KDM2A without influencing other KDMs.
Assuntos
Biocatálise , Proteínas F-Box , Histona Desmetilases com o Domínio Jumonji , Humanos , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Simulação de Dinâmica Molecular , Oxigênio/química , Oxigênio/metabolismo , Teoria QuânticaRESUMO
The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.
Assuntos
Aminoácidos , Bacillus subtilis , Dioxigenases , Ácidos Cetoglutáricos , Hidroxilação , Bacillus subtilis/enzimologia , Dioxigenases/metabolismo , Dioxigenases/química , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Aminoácidos/química , Aminoácidos/metabolismo , Estereoisomerismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Simulação de Dinâmica MolecularRESUMO
Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.
Assuntos
Catalase , Escherichia coli , Ácidos Cetoglutáricos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Escherichia coli/enzimologia , Catalase/metabolismo , Catalase/química , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/química , Streptomyces/enzimologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismoRESUMO
Epoxides, aziridines, and cyclopropanes are found in various medicinal natural products, including polyketides, terpenes, peptides, and alkaloids. Many classes of biosynthetic enzymes are involved in constructing these ring structures during their biosynthesis. This review summarizes our current knowledge regarding how α-ketoglutarate-dependent nonheme iron enzymes catalyze the formation of epoxides, aziridines, and cyclopropanes in nature, with a focus on enzyme mechanisms.
Assuntos
Aziridinas , Ferro , Ferro/química , Ácidos Cetoglutáricos/química , Catálise , Ciclopropanos , Compostos de EpóxiRESUMO
Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
Assuntos
Isocitrato Desidrogenase , Ácidos Cetoglutáricos , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Neoplasias/metabolismo , Especificidade por Substrato , Ligação Proteica/efeitos dos fármacos , CristalografiaRESUMO
This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII /2OG enzymes: a classâ 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line FeIII -OOâ - intermediate in PHF8 and the off-line FeIII -OOâ - intermediate in EFE.
Assuntos
Histona Desmetilases , Oxigenases , Ácidos Cetoglutáricos/química , Oxigênio , Compostos Férricos , Compostos Ferrosos/metabolismo , EtilenosRESUMO
Mononuclear non-heme Fe(II)- and α-ketoglutarate-dependent oxygenases (FeDOs) catalyze a site-selective C-H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to an FeDO facial triad variant, SadX, and the effects of that binding on HO⢠vs X⢠rebound. We establish not only that chloride and bromide enable non-native halogenation reactions but also that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3â¢, NCOâ¢, and OCN⢠rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO⢠and F⢠rebound from a putative Fe(III)(OH)(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO⢠and F⢠rebound transition states rather than an inaccessible barrier for F⢠rebound. Together, these results improve our understanding of the FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving these ligands, and inherent rebound preferences relative to HO⢠that will aid efforts to develop non-native catalysis using these enzymes.
Assuntos
Ácidos Cetoglutáricos , Oxigenases , Azidas , Cianatos , Compostos Férricos , Compostos Ferrosos/química , Fluoretos , Ácidos Cetoglutáricos/química , Ligantes , Oxigenases/metabolismoRESUMO
Fungal meroterpenoids are structurally diverse natural products with important biological activities. During their biosynthesis, α-ketoglutarate-dependent oxygenases (αKG-DOs) catalyze a wide range of chemically challenging transformation reactions, including desaturation, epoxidation, oxidative rearrangement, and endoperoxide formation, by selective C-H bond activation, to produce molecules with more complex and divergent structures. Investigations on the structure-function relationships of αKG-DO enzymes have revealed the intimate molecular bases of their catalytic versatility and reaction mechanisms. Notably, the catalytic repertoire of αKG-DOs is further expanded by only subtle changes in their active site and lid-like loop-region architectures. Owing to their remarkable biocatalytic potential, αKG-DOs are ideal candidates for future chemoenzymatic synthesis and enzyme engineering for the generation of terpenoids with diverse structures and biological activities.
Assuntos
Ácidos Cetoglutáricos , Oxigenases , Catálise , Compostos Ferrosos , Ácidos Cetoglutáricos/química , Oxirredução , Oxigenases/química , Oxigenases/metabolismoRESUMO
Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By using complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fueled biosynthetic processes while controlling the ratio of succinate/α-ketoglutarate (α-KG) to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells caused proliferation and survival defects when the TCA cycle was truncated, blocking carbon flux to support nucleoside biosynthesis. Replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. SDH deficiency induced a proinflammatory gene signature in T cells and promoted T helper 1 and T helper 17 lineage differentiation. An increasing succinate/α-KG ratio in SDH-deficient T cells promoted inflammation by changing the pattern of the transcriptional and chromatin accessibility signatures and consequentially increasing the expression of the transcription factor, PR domain zinc finger protein 1. Collectively, our studies revealed a role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.
Assuntos
Epigênese Genética , Succinato Desidrogenase , Proliferação de Células , Cromatina , Complexo II de Transporte de Elétrons/deficiência , Humanos , Inflamação/genética , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Erros Inatos do Metabolismo , Doenças Mitocondriais , Nucleosídeos , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , SuccinatosRESUMO
Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.
Assuntos
Proteínas Fúngicas/química , Ferro/química , Ácidos Cetoglutáricos/química , Oxirredutases N-Desmetilantes/química , Terpenos/química , Androsterona/química , Androsterona/metabolismo , Sítios de Ligação , Biocatálise , Cátions Bivalentes , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Humanos , Hidroxilação , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Moleculares , Mutação , Oxirredução , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Progesterona/química , Progesterona/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Terpenos/classificação , Terpenos/metabolismo , Testosterona/química , Testosterona/metabolismoRESUMO
Studies on the inhibition of the human 2-oxoglutarate dependent oxygenase JMJD6, which is a cancer target, by 2-oxoglutarate mimics / competitors, including human drugs, drug candidates, and metabolites relevant to cancer are described. JMJD6 assays employed NMR to monitor inhibitor binding and use of mass spectrometry to monitor JMJD6-catalysed lysine hydroxylation. Notably, some clinically applied prolyl hydroxylase inhibitors also inhibit JMJD6. The results will help enable the development of inhibitors selective for human oxygenases, including JMJD6.