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1.
Proc Natl Acad Sci U S A ; 121(13): e2321242121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38507448

RESUMO

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2, or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.


Assuntos
Ácidos Cicloexanocarboxílicos , Cicloexenos , Escherichia coli , Ubiquinona , Hidroxilação , Ubiquinona/metabolismo , Escherichia coli/metabolismo , Oxigênio/metabolismo
2.
Int J Biol Macromol ; 264(Pt 1): 130545, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38431000

RESUMO

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.


Assuntos
Ácidos Cumáricos , Oxigenases de Função Mista , Fenilacetatos , Pseudomonas aeruginosa , Oxigenases de Função Mista/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Hidroxilação , Escherichia coli/metabolismo
3.
Nat Commun ; 15(1): 2339, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38490987

RESUMO

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9α-hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1ß-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5α-ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.


Assuntos
Paclitaxel , Taxoides , Taxoides/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Hidroxilação , Oxirredução
4.
Amino Acids ; 56(1): 21, 2024 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-38461423

RESUMO

Metformin (N,N-dimethylbiguanide), an inhibitor of gluconeogenesis and insulin sensitizer, is widely used for the treatment of type 2 diabetes. In some patients with renal insufficiency, metformin can accumulate and cause lactic acidosis, known as metformin-associated lactic acidosis (MALA, defined as lactate ≥ 5 mM, pH < 7.35, and metformin concentration > 38.7 µM). Here, we report on the post-translational modification (PTM) of proline (Pro) to 4-hydroxyproline (OH-Pro) in metformin-associated lactic acidosis and in metformin-treated patients with Becker muscular dystrophy (BMD). Pro and OH-Pro were measured simultaneously by gas chromatography-mass spectrometry before, during, and after renal replacement therapy in a patient admitted to the intensive care unit (ICU) because of MALA. At admission to the ICU, plasma metformin concentration was 175 µM, with a corresponding lactate concentration of 20 mM and a blood pH of 7.1. Throughout ICU admission, the Pro concentration was lower compared to healthy controls. Renal excretion of OH-Pro was initially high and decreased over time. Moreover, during the first 12 h of ICU admission, OH-Pro seems to be renally secreted while thereafter, it was reabsorbed. Our results suggest that MALA is associated with hyper-hydroxyprolinuria due to elevated PTM of Pro to OH-Pro by prolyl-hydroxylase and/or inhibition of OH-Pro metabolism in the kidneys. In BMD patients, metformin, at the therapeutic dose of 3 × 500 mg per day for 6 weeks, increased the urinary excretion of OH-Pro suggesting elevation of Pro hydroxylation to OH-Pro. Our study suggests that metformin induces specifically the expression/activity of prolyl-hydroxylase in metformin intoxication and BMD.


Assuntos
Acidose Láctica , Diabetes Mellitus Tipo 2 , Metformina , Distrofia Muscular de Duchenne , Humanos , Metformina/efeitos adversos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Acidose Láctica/induzido quimicamente , Acidose Láctica/terapia , Hidroxiprolina , Cromatografia Gasosa-Espectrometria de Massas , Prolina , Hidroxilação , Distrofia Muscular de Duchenne/tratamento farmacológico , Ácido Láctico , Oxigenases de Função Mista/uso terapêutico , Hipoglicemiantes/efeitos adversos
5.
Xenobiotica ; 54(3): 124-137, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38358311

RESUMO

Nivasorexant was the first orexin-1 selective receptor antagonist entering clinical development. Despite encouraging preclinical evidence in animal models, a proof-of-concept trial in binge-eating patients recently failed to demonstrate its clinical utility in this population.Across species, nivasorexant clearance was driven by metabolism along seven distinct pathways, five of which were hydroxylation reactions in various locations of the molecule. The exact sites of metabolism were identified by means of mass spectrometry, the use of deuterated analogues, and finally confirmed by chemical references.CYP3A4 was the main cytochrome P450 enzyme involved in nivasorexant metabolism in vitro and accounting for about 90% of turnover in liver microsomes. Minor roles were taken by CYP2C9 and CYP2C19 but individually did not exceed 3-7%.In the rat, nivasorexant was mostly excreted via the bile after extensive metabolism, while urinary excretion was negligible. Only traces of the parent drug were detected in urine, bile, or faeces.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Humanos , Ratos , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Orexinas/metabolismo , Orexinas/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Citocromo P-450 CYP3A/metabolismo , Microssomos Hepáticos/metabolismo , Citocromo P-450 CYP2C19/metabolismo
6.
Crit Rev Toxicol ; 54(2): 92-122, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38363552

RESUMO

Polychlorinated biphenyls (PCBs) are persistent organic toxicants derived from legacy pollution sources and their formation as inadvertent byproducts of some current manufacturing processes. Metabolism of PCBs is often a critical component in their toxicity, and relevant metabolic pathways usually include their initial oxidation to form hydroxylated polychlorinated biphenyls (OH-PCBs). Subsequent sulfation of OH-PCBs was originally thought to be primarily a means of detoxication; however, there is strong evidence that it may also contribute to toxicities associated with PCBs and OH-PCBs. These contributions include either the direct interaction of PCB sulfates with receptors or their serving as a localized precursor for OH-PCBs. The formation of PCB sulfates is catalyzed by cytosolic sulfotransferases, and, when transported into the serum, these metabolites may be retained, taken up by other tissues, and subjected to hydrolysis catalyzed by intracellular sulfatase(s) to regenerate OH-PCBs. Dynamic cycling between PCB sulfates and OH-PCBs may lead to further metabolic activation of the resulting OH-PCBs. Ultimate toxic endpoints of such processes may include endocrine disruption, neurotoxicities, and many others that are associated with exposures to PCBs and OH-PCBs. This review highlights the current understanding of the complex roles that PCB sulfates can have in the toxicities of PCBs and OH-PCBs and research on the varied mechanisms that control these roles.


Assuntos
Bifenilos Policlorados , Bifenilos Policlorados/toxicidade , Bifenilos Policlorados/metabolismo , Hidroxilação , Sulfatos/toxicidade , Sulfatos/metabolismo , Poluição Ambiental , Substâncias Perigosas
7.
Commun Biol ; 7(1): 240, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38418569

RESUMO

Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.


Assuntos
Policitemia , Prolil Hidroxilases , Humanos , Prolil Hidroxilases/genética , Hidroxilação , Policitemia/genética , Mutação , Pró-Colágeno-Prolina Dioxigenase
8.
Int J Mol Sci ; 25(4)2024 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-38396831

RESUMO

In the present work, we report a neutral dinuclear copper(II) complex, [Cu2(L1)(OH)], derived from a new [N,O] donor Schiff base ligand L1 that was formed after the endogenous hydroxylation of an initial carbamate Schiff base H2L coordinated with copper ions in an electrochemical cell. The copper(II) complex has been fully characterized using different techniques, including X-ray diffraction. Direct current (DC) magnetic susceptibility measurements were also performed at variable temperatures, showing evidence of antiferromagnetic behavior. Its catalase-like activity was also tested, demonstrating that this activity is affected by temperature.


Assuntos
Cobre , Bases de Schiff , Cobre/química , Bases de Schiff/química , Hidroxilação , Ligantes , Catalase , Carbamatos , Cristalografia por Raios X
9.
Sci Rep ; 14(1): 3779, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38360875

RESUMO

Integral proteins or enzymes are still challenging to purify into their native state because of their need for an amphipathic environment and cofactors. Alkane hydroxylase (AlkB) is a membrane-bound enzyme that catalyzes the hydroxylation of a range of alkanes that have a broad spectrum of applications. In the current study, a novel approach has been explored for partial purification of alkane hydroxylase (AlkB) in its native state through restructuring the lipid bilayer of Penicillium chrysogenum SNP5 into a liposome to extend the native and protective environment to AlkB enzyme. Three different methods i.e., reverse-phase evaporation method (RPEM), detergent-based method (DBM), and ethanol injection method (EIM) have been used for reconstituting its native membrane into liposome. On characterizing liposomes through fluorescence imaging, AFM, and particle size analysis, the reverse-phase evaporation method gave the best results based on the size distribution (i.e., 100-300 nm), the morphology of liposomes, and maximum AlkB specific activity (i.e., 140.68 U/mg). The maximum reconstitution efficiency of 29.48% was observed in RPEM followed by 17.3% in DBM and 12.3% in EIM. On the characterization of the purified AlkB, the molecular weight was measured of 44.6 KDa and the thermostability of liposomes synthesized with the RPEM method was obtained maximum at 55 °C. This approach may open a new strategy for the purification of integral enzymes/proteins in their native state in the field of protein purification and its applications in diversified industries.


Assuntos
Alcanos , Lipossomos , Citocromo P-450 CYP4A/metabolismo , Hidroxilação , Alcanos/metabolismo
10.
Curr Opin Chem Biol ; 79: 102428, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38330792

RESUMO

The hypoxia-inducible factors are α,ß-heterodimeric transcription factors that mediate the chronic response to hypoxia in humans and other animals. Protein hydroxylases belonging to two different structural subfamilies of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase superfamily modify HIFα. HIFα prolyl-hydroxylation, as catalysed by the PHDs, regulates HIFα levels and, consequently, α,ß-HIF levels. HIFα asparaginyl-hydroxylation, as catalysed by factor inhibiting HIF (FIH), regulates the transcriptional activity of α,ß-HIF. The activities of the PHDs and FIH are regulated by O2 availability, enabling them to act as hypoxia sensors. We provide an overview of the biochemistry of the HIF hydroxylases, discussing evidence that their kinetic and structural properties may be tuned to their roles in the HIF system. Avenues for future research and therapeutic modulation are discussed.


Assuntos
Oxigenases de Função Mista , Fatores de Transcrição , Animais , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Fatores de Transcrição/metabolismo , Hipóxia/metabolismo , Hidroxilação
11.
EMBO J ; 43(6): 931-955, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38360997

RESUMO

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.


Assuntos
Proteína Beclina-1 , Carcinoma de Células Renais , Neoplasias Renais , Proteína Supressora de Tumor Von Hippel-Lindau , Animais , Humanos , Camundongos , Autofagia , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Hidroxilação , Neoplasias Renais/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
12.
Angew Chem Int Ed Engl ; 63(16): e202319624, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38376063

RESUMO

9,10-Secosteroids are an important group of marine steroids with diverse biological activities. Herein, we report a chemoenzymatic strategy for the concise, modular, and scalable synthesis of ten naturally occurring 9,10-secosteroids from readily available steroids in three to eight steps. The key feature lies in utilizing a Rieske oxygenase-like 3-ketosteroid 9α-hydroxylase (KSH) as the biocatalyst to achieve efficient C9-C10 bond cleavage and A-ring aromatization of tetracyclic steroids through 9α-hydroxylation and fragmentation. With synthesized 9,10-secosteroides, structure-activity relationship was evaluated based on bioassays in terms of previously unexplored anti-infective activity. This study provides experimental evidence to support the hypothesis that the biosynthetic pathway through which 9,10-secosteroids are formed in nature shares a similar 9α-hydroxylation and fragmentation cascade. In addition to the development of a biomimetic approach for 9,10-secosteroid synthesis, this study highlights the great potential of chemoenzymatic strategies in chemical synthesis.


Assuntos
Secoesteroides , Hidroxilação , Proteínas de Bactérias/metabolismo , Esteroides/química , Oxigenases de Função Mista/metabolismo
13.
Appl Environ Microbiol ; 90(3): e0225523, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38415602

RESUMO

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.


Assuntos
Oxigenases de Função Mista , Nicotina , Succinatos , Oxigenases de Função Mista/metabolismo , Nicotina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Hidroxilação , Piridinas/metabolismo
14.
Int J Mol Sci ; 25(2)2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38279222

RESUMO

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.


Assuntos
Produtos Biológicos , Oxigenases de Função Mista , Fenilacetatos , Oxigenases de Função Mista/metabolismo , Hidroxilação , Flavinas/química
15.
Nat Commun ; 15(1): 679, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38263179

RESUMO

Tetrodotoxin and congeners are specific voltage-gated sodium channel blockers that exhibit remarkable anesthetic and analgesic effects. Here, we present a scalable asymmetric syntheses of Tetrodotoxin and 9-epiTetrodotoxin from the abundant chemical feedstock furfuryl alcohol. The optically pure cyclohexane skeleton is assembled via a stereoselective Diels-Alder reaction. The dense heteroatom substituents are established sequentially by a series of functional group interconversions on highly oxygenated cyclohexane frameworks, including a chemoselective cyclic anhydride opening, and a decarboxylative hydroxylation. An innovative SmI2-mediated concurrent fragmentation, an oxo-bridge ring opening and ester reduction followed by an Upjohn dihydroxylation deliver the highly oxidized skeleton. Ruthenium-catalyzed oxidative alkyne cleavage and formation of the hemiaminal and orthoester under acidic conditions enable the rapid assembly of Tetrodotoxin, anhydro-Tetrodotoxin, 9-epiTetrodotoxin, and 9-epi lactone-Tetrodotoxin.


Assuntos
Cicloexanos , Estresse Oxidativo , Tetrodotoxina , Hidroxilação , Compostos Radiofarmacêuticos
16.
Chemistry ; 30(16): e202303845, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38212866

RESUMO

SznF, a member of the emerging family of heme-oxygenase-like (HO-like) di-iron oxidases and oxygenases, employs two distinct domains to catalyze the conversion of Nω-methyl-L-arginine (L-NMA) into N-nitroso-containing product, which can subsequently be transformed into streptozotocin. Using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP, we have mechanistically investigated the two sequential hydroxylations of L-NMA catalyzed by SznF's binuclear iron central domain. Mechanism B primarily involves the O-O bond dissociation, forming Fe(IV)=O, induced by the H+/e- introduction to the FeA side of µ-1,2-peroxo-Fe2(III/III), the substrate hydrogen abstraction by Fe(IV)=O, and the hydroxyl rebound to the substrate N radical. The stochastic addition of H+/e- to the FeB side (mechanism C) can transition to mechanism B, thereby preventing enzyme deactivation. Two other competing mechanisms, involving the direct O-O bond dissociation (mechanism A) and the addition of H2O as a co-substrate (mechanism D), have been ruled out.


Assuntos
Ferro , Oxigenases , Ferro/química , Hidroxilação , Estreptozocina , Oxigenases/química , Catálise
17.
Angew Chem Int Ed Engl ; 63(13): e202316133, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38279624

RESUMO

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.


Assuntos
Indenos , Oxigenases de Função Mista , Oxirredução , Hidroxilação , Biocatálise
18.
Steroids ; 204: 109362, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38278283

RESUMO

Research published between 2001 and 2022 on the functionalization of remote positions of steroids, as well as the use of this technique in the generation of biologically active compounds has been reviewed. In the first section of the analysis established and novel methods for activation of sites deemed to be remote were reported. A series of manganese- (mainly), rhodium-, ruthenium- and osmium-centered porphyrins as catalysts in the presence of PIDA as oxidant have effected hydroxylation at C-1, -5, -6, -7, -11, -14, -15, -16, -17, -20, -24 and -25. Dioxiranes have been utilized in inserting hydroxyl groups at the 5, 12, 14, 15, 16, 17, 20, 24 and 25 positions (tertiary centers for the most part). Alcohols at C-12 and -16 were oxidized further to ketones. The Schönecker oxidation, discovered and developed during the period, has revolutionized the selective functionalization at C-12 of steroids possessing a 17-keto group. In the presence of iron-centered PDP- and MCP-based catalysts, hydrogen peroxide and acetic acid, substrates tended to be hydroxylated at C-6 and -12, with further oxidation to ketones often accompanying this reaction. The hypohalite reaction, utilizing the more modern Suarez conditions (irradiation in the presence of iodine and PIDA), was reported to facilitate the insertion of a hydroxyl moiety five atoms away from an existing alcohol oxygen. Steroidal-3ß-diazoacetates tend to decompose on heating with di-rhodium-centered catalysts while activating carbons four or five atoms away. Chromium- and iron-based acetates were observed to functionalize C-5 and -25. Other reactions involving ring cleavage and halogenation, ketone irradiation and α-hydroxylation of ethers were also covered. The syntheses of compounds with marked biological activity from readily available steroids is described in the second section of the study. Cyclopamine, cephalostatin-1, ritterazine B and three polyhydroxypregnanaes (pergularin, utendin and tomentogenin) were generated in sequences in which a key step required hydroxylation at C-12 using the Schönecker reaction. A crucial stage in the preparation of cortistatin A, the saundersioside core, eurysterol A, 5,6-dihydroglaucogenin C, as well as clinostatins A and B involved the functionalization of C-18 or -19 utilizing hypohalite chemistry. The synthetic route to xestobergsterol A, pavonin-4-aglycone and ouagabagenin included a transformation where ketone irradiation played a part in either producing a Δ14 or a C-19 activated steroid. The radical relay reaction, where a 17α-chloro-steroid was formed, was central in the generation of pythocholic acid. The lead tetraacetate reaction was pivotal in the functionalization of C-19 during the synthesis of cyclocitrinol.


Assuntos
Ródio , Ródio/química , Esteroides/química , Hidroxilação , Álcoois , Cetonas , Ferro , Catálise
19.
PeerJ ; 12: e16711, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38188169

RESUMO

Polymethoxylated flavonoids (PMFs) are a subclass of flavonoids found in citrus fruits that have shown multifunctional biological activities and potential anticancer effects against breast cancer. We studied the absorption, metabolism, species source, toxicity, anti-cancer mechanisms, and molecular targets of PMFs to better utilize their anticancer activity against breast cancer. We discuss the absorption and metabolism of PMFs in the body, including the methylation, demethylation, and hydroxylation processes. The anticancer mechanisms of PMFs against breast cancer were also reviewed, including the estrogen activity, cytochrome P-450 enzyme system, and arylhydrocarbon receptor (AhR) inhibition, along with various molecular targets and potential anticancer effects. Although PMFs may be advantageous in the prevention and treatment for breast cancer, there is a lack of clinical evidence and data to support their efficacy. Despite their promise, there is still a long way to go before PMFs can be applied clinically.


Assuntos
Citrus , Neoplasias , Frutas , Estrogênios , Hidroxilação , Flavonoides/farmacologia
20.
Yakugaku Zasshi ; 144(2): 197-202, 2024.
Artigo em Japonês | MEDLINE | ID: mdl-38296497

RESUMO

My research focused on the effects of various drugs on (1) dopamine formation from p-tyramine catalyzed by polymorphic cytochrome P450 (CYP or P450) 2D6 variants and (2) endogenous steroid hormone hydroxylation catalyzed by CYP3A subfamily members (CYP3A4, CYP3A5, CYP3A7). The activation (cooperativity) of metabolic reactions catalyzed by P450s was especially emphasized. The effects of various psychotropic agents on dopamine formation from p-tyramine, catalyzed by wild-type CYP2D6.1 and CYP2D6 variants, including CYP2D6.2 (Arg296Cys;Ser486Thr), CYP2D6.10 (Pro34Ser;Ser486Thr), and CYP2D6.39 (Ser486Thr) were compared. Michaelis (Km) and inhibition (Ki) constants of the psychotropic agents in the presence of CYP2D6.10 were higher than those observed in the presence of other CYP2D6 variants. Fluvoxamine, fluoxetine, milnacipran, and haloperidol activated CYP2D6-catalyzed dopamine formation [decreasing the Km and/or increasing the maximal velocity (kcat)], and this activation was CYP2D6 variant-dependent. Regarding the CYP3A subfamily, the effects of various compounds including endogenous steroid hormones on the 6ß-hydroxylation of steroid hormones, such as testosterone, progesterone, and cortisol, were determined; it was found that testosterone, dehydroepiandrosterone, and/or α-naphthoflavone activated 6ß-hydroxylation of cortisol and/or progesterone, but the effects varied in the presence of different CYP3A subfamily members. Further studies are required to confirm the mechanisms and therapeutic relevance of these activation phenomena.


Assuntos
Citocromo P-450 CYP2D6 , Progesterona , Humanos , Citocromo P-450 CYP2D6/metabolismo , Progesterona/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hidrocortisona/metabolismo , Dopamina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Esteroides/metabolismo , Hidroxilação , Tiramina/metabolismo , Testosterona/metabolismo , Catálise , Microssomos Hepáticos/metabolismo
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