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1.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445153

RESUMO

In order to achieve a desired therapeutic effect in schizophrenia patients and to maintain their mental wellbeing, pharmacological therapy needs to be continued for a long time, usually from the onset of symptoms and for the rest of the patients' lives. The aim of our present research is to find out the in vivo effect of chronic treatment with atypical neuroleptic iloperidone on the expression and activity of cytochrome P450 (CYP) in rat liver. Male Wistar rats received a once-daily intraperitoneal injection of iloperidone (1 mg/kg) for a period of two weeks. Twenty-four hours after the last dose, livers were excised to study cytochrome P450 expression (mRNA and protein) and activity, pituitaries were isolated to determine growth hormone-releasing hormone (GHRH), and blood was collected for measuring serum concentrations of hormones and interleukin. The results showed a broad spectrum of changes in the expression and activity of liver CYP enzymes, which are important for drug metabolism (CYP1A, CYP2B, CYP2C, and CYP3A) and xenobiotic toxicity (CYP2E1). Iloperidone decreased the expression and activity of CYP1A2, CP2B1/2, CYP2C11, and CYP3A1/2 enzymes but increased that of CYP2E1. The CYP2C6 enzyme remained unchanged. At the same time, the level of GHRH, GH, and corticosterone decreased while that of T3 increased, with no changes in IL-2 and IL-6. The presented results indicate neuroendocrine regulation of the investigated CYP enzymes during chronic iloperidone treatment and suggest a possibility of pharmacokinetic/metabolic interactions produced by the neuroleptic during prolonged combined treatment with drugs that are substrates of iloperidone-affected CYP enzymes.


Assuntos
Antipsicóticos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Isoxazóis/farmacologia , Fígado/efeitos dos fármacos , Piperidinas/farmacologia , Animais , Antipsicóticos/administração & dosagem , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica/efeitos dos fármacos , Isoxazóis/administração & dosagem , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Piperidinas/administração & dosagem , Ratos , Ratos Wistar , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo
2.
Molecules ; 26(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361831

RESUMO

The interaction of small organic molecules such as drugs, agrochemicals, and cosmetics with cytochrome P450 enzymes (CYPs) can lead to substantial changes in the bioavailability of active substances and hence consequences with respect to pharmacological efficacy and toxicity. Therefore, efficient means of predicting the interactions of small organic molecules with CYPs are of high importance to a host of different industries. In this work, we present a new set of machine learning models for the classification of xenobiotics into substrates and non-substrates of nine human CYP isozymes: CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. The models are trained on an extended, high-quality collection of known substrates and non-substrates and have been subjected to thorough validation. Our results show that the models yield competitive performance and are favorable for the detection of CYP substrates. In particular, a new consensus model reached high performance, with Matthews correlation coefficients (MCCs) between 0.45 (CYP2C8) and 0.85 (CYP3A4), although at the cost of coverage. The best models presented in this work are accessible free of charge via the "CYPstrate" module of the New E-Resource for Drug Discovery (NERDD).


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Aprendizado de Máquina , Xenobióticos/classificação , Xenobióticos/metabolismo , Animais , Humanos , Especificidade por Substrato
3.
Toxicol Lett ; 350: 267-282, 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34352333

RESUMO

The open source database "OpenCYP database" has been developed based on the results of extensive literature searches from the peer-reviewed literature. OpenCYP provides data on human variability on baseline of activities and polymophism frequencies for selected cytochrome P-450 isoforms (CYP1A2, CYP2A6, CYP2D6, CYP3A4/3A5 and CYP3A7) in healthy adult populations from world populations. CYP enzymatic activities were generally expressed as the metabolic ratio (MR) between an unchanged probe drug and its metabolite(s) in urine or plasma measured in healthy adults. Data on other age groups were very limited and fragmented, constituting an important data gap. Quantitative comparisons were often hampered by the different experimental conditions used. However, variability was quite limited for CYP1A2, using caffeine as a probe substrate, with a symmetrical distribution of metabolic activity values. For CYP3A4, human variability was dependent on the probe substrate itself with low variability when data considering the dextromethorphan/demethilathed metabolite MR were used and large variability when the urinary 6ß-hydroxycortisol/cortisol ratio was used. The largest variability in CYP activity was shown for CYP2D6 activity, after oral dosing of dextromethorphan, for which genetic polymorphisms are well characterised and constitute a significant source of variability. It is foreseen that the OpenCYP database can contribute to promising tools to support the further development of QIVIVE and PBK models for human risk assessment of chemicals particularly when combined with information on isoform-specific content in cells using proteomic approaches.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bases de Dados Genéticas , Polimorfismo Genético , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância da População , Proteômica
4.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360577

RESUMO

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s' interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.


Assuntos
Aminoácidos/metabolismo , Azóis/metabolismo , Basidiomycota/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fluconazol/metabolismo , Proteínas Fúngicas/metabolismo , Itraconazol/metabolismo , Aminoácidos/química , Antifúngicos/química , Antifúngicos/metabolismo , Azóis/química , Simulação por Computador , Sistema Enzimático do Citocromo P-450/química , Fluconazol/química , Proteínas Fúngicas/química , Itraconazol/química , Ligantes , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
5.
Xenobiotica ; 51(9): 995-1009, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34224301

RESUMO

Nine forms of recombinant cytochrome P450 (P450 or CYP) enzymes were used to study roles of individual P450 enzymes in the oxidation of flavone and some other flavonoids, 4'-hydroxyflavone and 4'-, 3'-, and 2'-methoxyflavones, by human liver microsomes using LC-MS/MS analysis.As has been reported previously , 4'-, 3'-, and 2'-methoxyflavones were preferentially O-demethylated by human liver P450 enzymes to form 4'-, 3'-, and 2'-hydroxylated flavones and also 3',4'-dihydroxyflavone from the former two substrates.In comparisons of product formation by oxidation of these methoxylated flavones, CYP2A6 was found to be a major enzyme catalysing flavone 4'- and 3'-hydroxylations by human liver microsomes but did not play significant roles in 2'-hydroxylation of flavone, O-demethylations of three methoxylated flavones, and the oxidation of 4'-hydroxyflavone to 3',4'-dihydroxyflavone.The effects of anti-CYP2A6 IgG and chemical P450 inhibitors suggested that different P450 enzymes, as well as CYP2A6, catalysed oxidation of these flavonoids at different positions by liver microsomes.These studies suggest that CYP2A6 catalyses flavone 4'- and 3'-hydroxylations in human liver microsomes and that other P450 enzymes have different roles in oxidizing these flavonoids.


Assuntos
Flavonas , Microssomos Hepáticos , Cromatografia Líquida , Citocromo P-450 CYP2A6/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/metabolismo , Flavonoides/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Oxirredução , Espectrometria de Massas em Tandem
6.
Xenobiotica ; 51(9): 1060-1070, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34330191

RESUMO

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.


Assuntos
Aldeído Oxidase , Microssomos Hepáticos , Aldeído Oxidase/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Taxa de Depuração Metabólica , Camundongos , Microssomos Hepáticos/metabolismo , Especificidade da Espécie
7.
Methods Mol Biol ; 2342: 29-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272690

RESUMO

Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Algoritmos , Ligação Competitiva , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/farmacologia , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Cinética
8.
Methods Mol Biol ; 2342: 89-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272692

RESUMO

Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYPs) enzymes are catalytically promiscuous and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appear to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein, is a discussion of two distinct type of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ferro/metabolismo , Algoritmos , Animais , Catálise , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Cinética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Oxirredução
9.
Methods Mol Biol ; 2342: 51-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272691

RESUMO

This chapter describes the types of irreversible inhibition of drug-metabolizing enzymes and the methods commonly employed to quantify the irreversible inhibition and subsequently predict the extent and time course of clinically important drug-drug interactions.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Preparações Farmacêuticas/química , Catálise , Inibidores das Enzimas do Citocromo P-450/farmacologia , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Cinética , Fatores de Tempo , Xenobióticos/farmacologia
10.
Methods Mol Biol ; 2342: 171-192, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272695

RESUMO

The complex enzyme kinetics displayed by drug-metabolizing cytochrome P450 enzymes (CYPs) (see Chapter 9 ) can, in part, be explained by an examination of their crystallographic protein structures. Fortunately, despite low sequence similarity between different families of drug-metabolizing CYPs, there exists a high degree of structural homology within the superfamily. This similarity in the protein fold allows for a direct comparison of the structural features of CYPs that contribute toward differences in substrate binding, heterotropic and homotropic cooperativity, and genetic variability in drug metabolism. In this chapter, we first provide an overview of the nomenclature and the role of structural features that are common in all CYPs. We then apply these definitions to understand the different substrate specificities and functions in the CYP3A, CYP2C, and CYP2D families of enzymes.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Variação Genética , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Inativação Metabólica , Cinética , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína , Especificidade por Substrato
11.
Methods Mol Biol ; 2342: 237-256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272697

RESUMO

The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ativação Metabólica , Algoritmos , Domínio Catalítico , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estresse Oxidativo , Especificidade por Substrato , Xenobióticos/farmacocinética
12.
Methods Mol Biol ; 2342: 595-629, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272707

RESUMO

This chapter provides regulatory perspectives on how to translate in vitro drug metabolism findings into in vivo drug-drug interaction (DDI) predictions and how this affects the decision of conducting in vivo DDI evaluation. The chapter delineates rationale and analyses that have supported the recommendations in the U.S. Food and Drug Administration (FDA) DDI guidances in terms of in vitro-in vivo extrapolation of cytochrome P450 (CYP) inhibition-mediated DDI potential for investigational new drugs and their metabolites as substrates or inhibitors. The chapter also describes the framework and considerations to assess UDP-glucuronosyltransferase (UGT) inhibition-mediated DDI potential for drugs as substrates or inhibitors. The limitations of decision criteria and further improvements needed are also discussed. Case examples are provided throughout the chapter to illustrate how decision criteria have been utilized to evaluate in vivo DDI potential from in vitro data.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/metabolismo , Legislação de Medicamentos/organização & administração , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Interações Medicamentosas , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/química , Humanos , Cinética , Guias de Prática Clínica como Assunto , Estados Unidos , United States Food and Drug Administration/legislação & jurisprudência
13.
Methods Mol Biol ; 2342: 665-684, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272711

RESUMO

An appreciation of enzyme kinetic principles can be applied in a number of drug metabolism applications. The concept for this chapter arose from a simple discussion on selecting appropriate time points to most efficiently assess metabolite profiles in a human Phase 1a clinical study (Subheading 4). By considering enzyme kinetics, a logical approach to the issue was derived. The dialog was an important learning opportunity for the participants in the discussion, and we have endeavored to capture this experience with other questions related to determination of Km and Vmax parameters, a consideration of the value of hepatocytes vs. liver microsomes, and enzyme inhibition parameters.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Metabolômica/métodos , Preparações Farmacêuticas/administração & dosagem , Algoritmos , Ensaios Clínicos Fase I como Assunto , Cálculos da Dosagem de Medicamento , Humanos , Cinética , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/metabolismo
14.
Methods Mol Biol ; 2342: 809-823, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272718

RESUMO

Often it may be convenient and efficient to address multiple research questions with a single experiment. In many instances, however, the best approach is to design the experiment to address one question at a time. The design of enzyme mapping experiments is discussed in this chapter, focusing on considerations pertinent to the study of aldehyde oxidase (AO) vs. cytochrome P450 metabolism. Specifically, a case is presented in which reduced glutathione (GSH) was included in an experiment with human liver S9 fraction to trap reactive metabolites generated from cytochrome P450-mediated metabolism of lapatinib and its O-dealkylated metabolite, M1 (question 1). The AO inhibitor hydralazine was included in this experiment to investigate the involvement of AO-mediated metabolism of M1 (question 2). The presence of GSH was found to interfere with the inhibitory activity of hydralazine. Consideration of the time-dependent nature of hydralazine inhibitory activity toward AO when designing this experiment could have predicted the potential for GSH to interfere with hydralazine. This case underscores the importance of clearly identifying the research question, tailoring the experimental protocol to answer that question, and then meticulously considering how the experimental conditions could influence the results, particularly if attempting to address multiple questions with a single experiment.


Assuntos
Aldeído Oxidase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/metabolismo , Hidralazina/farmacocinética , Lapatinib/farmacocinética , Ativação Metabólica , Interações Medicamentosas , Hepatócitos/citologia , Humanos , Microssomos Hepáticos/enzimologia , Oxirredução , Projetos de Pesquisa , Fatores de Tempo
15.
J Chem Phys ; 155(2): 025101, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34266281

RESUMO

The function of an enzyme depends on its dynamic structure, and the catalytic mechanism has long been an active focus of research. The principle for interpreting protein selectivity and fidelity stems from optimization of the active site upon protein-substrate complexation, i.e., a lock-and-key configuration, on which most protein-substrate molecule binding recognition, and hence drug discovery, relies. Yet another thought has been to incorporate the protein folding interior tunnels for stereo- and regio-selectivity along the protein-substrate or protein-ligand/inhibitor binding process. Free energy calculations provide valuable information for molecular recognition and protein-ligand binding dynamics and kinetics. In this study, we focused on the kinetics of cytochrome P450 proteins (CYP450s) and the protein interior tunnel structure-dynamics relationship in terms of the substrate binding and leaving mechanism. A case in point is given by the prostaglandin H2 (PGH2) homologous isomerase of prostacyclin synthase. To calculate the reactant and product traversing the tunnels to and from the heme site, the free energy paths and tunnel potentials of mean force are constructed from steered molecular dynamics simulations and adaptive basing force umbrella sampling simulations. We explore the binding tunnels and critical residue lining characteristics for the ligand traverse and the underlying mechanism of CYP450 activity. Our theoretical analysis provides insights into the decisive role of the substrate tunnel binding process of the CYP450 mechanism and may be useful in drug design and protein engineering contexts.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica
16.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281173

RESUMO

Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.


Assuntos
Neoplasias da Mama/metabolismo , Epóxido Hidrolases/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Epóxido Hidrolases/genética , Compostos de Epóxi/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Deleção de Genes , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo
17.
Int J Mol Sci ; 22(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34281222

RESUMO

There are two types of cytochrome P450 enzymes in nature, namely, the monooxygenases and the peroxygenases. Both enzyme classes participate in substrate biodegradation or biosynthesis reactions in nature, but the P450 monooxygenases use dioxygen, while the peroxygenases take H2O2 in their catalytic cycle instead. By contrast to the P450 monooxygenases, the P450 peroxygenases do not require an external redox partner to deliver electrons during the catalytic cycle, and also no external proton source is needed. Therefore, they are fully self-sufficient, which affords them opportunities in biotechnological applications. One specific P450 peroxygenase, namely, P450 OleTJE, reacts with long-chain linear fatty acids through oxidative decarboxylation to form hydrocarbons and, as such, has been implicated as a suitable source for the biosynthesis of biofuels. Unfortunately, the reactions were shown to produce a considerable amount of side products originating from Cα and Cß hydroxylation and desaturation. These product distributions were found to be strongly dependent on whether the substrate had substituents on the Cα and/or Cß atoms. To understand the bifurcation pathways of substrate activation by P450 OleTJE leading to decarboxylation, Cα hydroxylation, Cß hydroxylation and Cα-Cß desaturation, we performed a computational study using 3-phenylpropionate and 2-phenylbutyrate as substrates. We set up large cluster models containing the heme, the substrate and the key features of the substrate binding pocket and calculated (using density functional theory) the pathways leading to the four possible products. This work predicts that the two substrates will react with different reaction rates due to accessibility differences of the substrates to the active oxidant, and, as a consequence, these two substrates will also generate different products. This work explains how the substrate binding pocket of P450 OleTJE guides a reaction to a chemoselectivity.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Modelos Químicos , Fenilbutiratos/metabolismo , Fenilpropionatos/metabolismo
18.
Molecules ; 26(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204747

RESUMO

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Hydrogenophilaceae/metabolismo , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Sistema Enzimático do Citocromo P-450/fisiologia , Hydrogenophilaceae/enzimologia , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologia
20.
J Phys Chem B ; 125(30): 8419-8430, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34313131

RESUMO

Spirooxindoles are pivotal biofunctional groups widely distributed in natural products and clinic drugs. However, construction of such subtle chiral skeletons is a long-standing challenge to both organic and bioengineering scientists. The knowledge of enzymatic spirooxindole formation in nature may inspire rational design of new catalysts. To this end, we presented a theoretical investigation on the elusive mechanism of the spiro-ring formation at the 3-position of oxindole mediated by cytochrome P450 enzymes (P450). Our calculated results demonstrated that the electrophilic attack of CpdI, the active species of P450, to the substrate, shows regioselectivity, i.e., the attack at the C9 position forms a tetrahedral intermediate involving an unusual feasible charge-shift C9δ+-Oδ- bond, while the attack at the C1 position forms an epoxide intermediate. The predominant route is the first route with the charge-shift bonding intermediate due to holding a relatively lower barrier by >5 kcal mol-1 than the epoxide route, which fits the experimental observations. Such a delocalized charge-shift bond facilitates the formation of a spiro-ring mainly through elongation of the C1-C9 bond to eliminate the aromatization of the tricyclic beta-carboline. Our theoretical results shed profound mechanistic insights for the first time into the elusive spirooxindole formation mediated by P450s.


Assuntos
Sistema Enzimático do Citocromo P-450 , Compostos de Epóxi , Catálise , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução
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