RESUMO
Dihydropyrimidine dehydrogenase (DPD) is an enzyme that uses an elaborate architecture to catalyze a simple net reaction: the reduction of the vinylic bond of uracil and thymine. Known DPDs have two active sites separated by approximately 60 Å. One active site has an FAD cofactor and binds NAD(P) and the other has an FMN cofactor and binds pyrimidines. The intervening distance is spanned by four Fe4S4 centers that act as an electron conduit. Recent advancements with porcine DPD have revealed unexpected chemical sequences where the enzyme undergoes reductive activation by transferring two electrons from NADPH to the FMN via the FAD such that the active form has the cofactor set FADâ¢4(Fe4S4)â¢FMNH2. Here we describe the first comprehensive kinetic investigation of a bacterial form of DPD. Using primarily transient state methods, DPD from E. coli (EcDPD) was shown to have a similar mechanism to that observed with the mammalian form in that EcDPD is observed to undergo reductive activation before pyrimidine reduction and displays half-of-sites activity. However, two distinct aspects of the EcDPD reaction relative to the mammalian enzyme were observed that relate to the effector roles for substrates: (i) the enzyme will rapidly take up electrons from NADH, reducing a flavin in the absence of pyrimidine substrate, and (ii) the activated form of the enzyme can become fully oxidized by transferring electrons to pyrimidine substrates in the absence of NADH.
Assuntos
Escherichia coli , NAD , Suínos , Animais , NAD/metabolismo , Di-Hidrouracila Desidrogenase (NADP)/química , Cinética , Uracila , Mamíferos/metabolismo , Oxirredução , Flavina-Adenina Dinucleotídeo/químicaRESUMO
Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by â¼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe4S4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe4S4 centers to yield the active state with a cofactor set of FADâ¢4(Fe4S4)â¢FMNH2. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP+ indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzymeâ¢pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP+ DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.
Assuntos
Di-Hidrouracila Desidrogenase (NADP) , Pirimidinas , Animais , Oxirredução , Di-Hidrouracila Desidrogenase (NADP)/química , NADP/metabolismo , Espectrofotometria , Pirimidinas/metabolismo , Cinética , Flavina-Adenina Dinucleotídeo/química , Mamíferos/metabolismoRESUMO
Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the behavior of the enzyme had remained elusive. It was recently shown that the active form of DPD has the cofactor set of FAD-4(Fe4S4)-FMNH2. This two-electron reduced state is consistent with fewer mechanistic possibilities and data suggests that the instigating and rate determining step in the catalytic cycle is reduction of the pyrimidine substrate that is followed by relatively rapid oxidation of NADPH at the FAD that, via the electron conduit of the 4(Fe4S4) centers, reinstates the FMNH2 cofactor for subsequent catalytic turnover.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Animais , Catálise , Di-Hidrouracila Desidrogenase (NADP)/química , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Hidroquinonas/metabolismo , Mamíferos , Oxirredução , Conformação Proteica , Domínios Proteicos , Especificidade por SubstratoRESUMO
The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by â¼60 Å that are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Hidrogênio/metabolismo , Animais , Domínio Catalítico , Di-Hidrouracila Desidrogenase (NADP)/fisiologia , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Hidrogênio/química , Cinética , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Espectrofotometria/métodos , SuínosRESUMO
Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of â¼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 Å across the protein.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Uracila/análogos & derivados , Animais , Domínio Catalítico , Di-Hidrouracila Desidrogenase (NADP)/química , Sinergismo Farmacológico , Ligação Proteica , Suínos , Uracila/metabolismo , Uracila/farmacologiaRESUMO
PURPOSE: Several clinical guidelines recommend genetic screening of DPYD, including coverage of the variants c.1905 + 1G>A(DPYD*2A), c.1679T>G(DPYD*13), c.2846A>T, and c.1129-5923C>G, before initiating treatment with fluoropyrimidines. However, this screening is often inadequate at predicting the occurrence of severe fluoropyrimidine-induced toxicity in patients. METHODS: Using a complementary approach combining whole DPYD exome sequencing and in silico and structural analysis, as well as phenotyping of DPD by measuring uracilemia (U), dihydrouracilemia (UH2), and the UH2/U ratio in plasma, we were able to characterize and interpret DPYD variants in 28 patients with severe fluoropyrimidine-induced toxicity after negative screening. RESULTS: Twenty-five out of 28 patients (90%) had at least 1 variant in the DPYD coding sequence, and 42% of the variants (6/14) were classified as potentially deleterious by at least 2 of the following algorithms: SIFT, Poly-Phen-2, and DPYD varifier. We identified two very rare deleterious mutations, namely, c.2087G>A (p.R696H) and c.2324T>G (p.L775W). We were able to demonstrate partial DPD deficiency, as measured by the UH2/U ratio in a patient carrying the variant p.L775W for the first time. CONCLUSION: Whole exon sequencing of DPYD in patients with suspicion of partial DPD deficiency can help to identify rare or new variants that lead to enzyme inactivation. Combining different techniques can yield abundant information without increasing workload and cost burden, thus making it a useful approach for implementation in patient care.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Deficiência da Di-Hidropirimidina Desidrogenase/induzido quimicamente , Deficiência da Di-Hidropirimidina Desidrogenase/genética , Di-Hidrouracila Desidrogenase (NADP)/genética , Variantes Farmacogenômicos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Capecitabina/efeitos adversos , Simulação por Computador , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Uracila/análogos & derivados , Uracila/sangueRESUMO
Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation. Spectrophotometric deconvolution suggests that these electrons reside on one of the two flavins. The fact that oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed, indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from the C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and high-performance liquid chromatography product analysis of single-turnover reactions with limiting NADPH confirmed a 2:1 NADPH:pyrimidine stoichiometry for the enzyme, accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/química , Mononucleotídeo de Flavina/química , NADP/química , Pirimidinas/química , Sus scrofa , Animais , Di-Hidrouracila Desidrogenase (NADP)/genética , Ativação EnzimáticaRESUMO
Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of uracil and thymine bases with electrons derived from NADPH. The mammalian DPD enzyme is a functional homodimer and has an elaborate cofactor arrangement. Two flavin cofactors (FAD and FMN) reside in two active site cavities that are separated by around 60 Å. The flavins are apparently bridged by four Fe4S4 clusters, two of which are provided by the partner protomer of the dimer. The study of DPD has been hampered by modest yield from both native sources and from heterologous expression in E. coli. In addition, minimal active enzyme is obtained when the DPD gene is fused to an N-terminal 6His-tag. This limitation has dictated the use of traditional purification methods that are made more challenging by apparent over-expression of truncated and/or non-active forms of DPD. Here we detail methods of expression and purification that result in a ~4-fold improvement in the yield of active porcine DPD when expressed in E. coli BL21 DE3 cells via the pET plasmid expression system. The addition of ferrous ions and sulfate during induction provide a small increase in purified active enzyme. However, the addition of FAD and FMN during cell lysis results in a substantial increase in activity that also reduces the relative proportion of non-active, high molecular weight protein contaminants. We also describe methods that permit correlation of the flavin content with the amount of active enzyme and thus permit simple, rapid quantitation and evaluation of purified DPD sample.
Assuntos
Di-Hidrouracila Desidrogenase (NADP) , Expressão Gênica , Proteínas Recombinantes de Fusão , Animais , Di-Hidrouracila Desidrogenase (NADP)/biossíntese , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Suínos/genéticaRESUMO
5-fluorouracil (5-FU) is an anticancer drug, which inhibits human thymidine phosphorylase (hTP) and plays a key role in maintaining the process of DNA replication and repair. It is involved in regulating pyrimidine nucleotide production, by which it inhibits the mechanism of cell proliferation and cancerous tumor growth. However, up to 80% of the administered drug is metabolized by dihydropyrimidine dehydrogenase (DPD). This work compares binding of 5-FU and its analogues to hTP and DPD, and suggests strategies to reduce drug binding to DPD to decrease the required dose of 5-FU. An important feature between the proteins studied here was the difference of charge distribution in their binding sites, which can be exploited for designing drugs to selectively bind to the hTP. The 5-FU presence was thought to be required for a closed conformation. Comparison of the calculation results pertaining to unliganded and liganded protein showed that hTP could still undergo open-closed conformations in the absence of the ligand; however, the presence of a positively charged ligand better stabilizes the closed conformation and rigidifies the core region of the protein more than unliganded or neutral liganded system. The study has also shown that one of the three hinge segments linking the two major α and α/ß domains of the hTP is an important contributing factor to the enzyme's open-close conformational twist during its inactivation-activation process. In addition, the angle between the α/ß-domain and the α-domain has shown to undergo wide rotations over the course of MD simulation in the absence of a phosphate, suggesting that it contributes to the stabilization of the closed conformation of the hTP.
Assuntos
Antimetabólitos Antineoplásicos/química , Di-Hidrouracila Desidrogenase (NADP)/química , Fluoruracila/química , Bibliotecas de Moléculas Pequenas/química , Timidina Fosforilase/química , Sítios de Ligação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5-fluorouracil (5-FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no-synonymous DPYD variants. We developed a DPYD-specific variant classifier (DPYD-Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5-FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD-Varifier have great potential to individualize medicine and improve the clinical decision-making process.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Simulação por Computador , Di-Hidrouracila Desidrogenase (NADP)/genética , Fluoruracila/toxicidade , Aprendizado de Máquina , Mutação de Sentido Incorreto , Farmacogenética/métodos , Testes Farmacogenômicos/métodos , Variantes Farmacogenômicos , Antimetabólitos Antineoplásicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Relação Dose-Resposta a Droga , Fluoruracila/metabolismo , Frequência do Gene , Genótipo , Células HCT116 , Células HEK293 , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Valor Preditivo dos Testes , Conformação Proteica , Medição de Risco , Relação Estrutura-AtividadeRESUMO
UNLABELLED: New strategies are needed to diagnose and target human melanoma. To this end, genomic analyses was performed to assess somatic mutations and gene expression signatures using a large cohort of human skin cutaneous melanoma (SKCM) patients from The Cancer Genome Atlas (TCGA) project to identify critical differences between primary and metastatic tumors. Interestingly, pyrimidine metabolism is one of the major pathways to be significantly enriched and deregulated at the transcriptional level in melanoma progression. In addition, dihydropyrimidine dehydrogenase (DPYD) and other important pyrimidine-related genes: DPYS, AK9, CAD, CANT1, ENTPD1, NME6, NT5C1A, POLE, POLQ, POLR3B, PRIM2, REV3L, and UPP2 are significantly enriched in somatic mutations relative to the background mutation rate. Structural analysis of the DPYD protein dimer reveals a potential hotspot of recurring somatic mutations in the ligand-binding sites as well as the interfaces of protein domains that mediated electron transfer. Somatic mutations of DPYD are associated with upregulation of pyrimidine degradation, nucleotide synthesis, and nucleic acid processing while salvage and nucleotide conversion is downregulated in TCGA SKCM. IMPLICATIONS: At a systems biology level, somatic mutations of DPYD cause a switch in pyrimidine metabolism and promote gene expression of pyrimidine enzymes toward malignant progression.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/genética , Melanoma/genética , Mutação , Pirimidinas/metabolismo , Neoplasias Cutâneas/genética , Sítios de Ligação , Di-Hidrouracila Desidrogenase (NADP)/química , Progressão da Doença , Humanos , Modelos Moleculares , Biologia de SistemasRESUMO
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, P = 0.025; 116%, P = 0.049; 130%, P = 0.0077; 138%, P = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Antimetabólitos Antineoplásicos/química , Biotransformação , Di-Hidrouracila Desidrogenase (NADP)/biossíntese , Di-Hidrouracila Desidrogenase (NADP)/química , Ensaios Enzimáticos , Fluoruracila/química , Frequência do Gene , Estudos de Associação Genética , Células HEK293 , Humanos , Dados de Sequência MolecularRESUMO
OBJECTIVES: Evaluation of a non-synonymous mutation associated with dihydropyrimidine dehydrogenase (DPD) deficiency. DESIGN AND METHODS: DPD enzyme analysis, mutation analysis and molecular dynamics simulations based on the 3D-model of DPD. RESULTS: The substitution Lys63Glu is likely to affect the FAD binding pocket within the DPD protein and contributes to a near-complete DPD deficiency in a patient with developmental retardation. CONCLUSIONS: Like other DPD variants attenuating FAD binding, Lys63Glu should be included in screening for DPD deficiency.
Assuntos
Deficiência da Di-Hidropirimidina Desidrogenase/urina , Di-Hidrouracila Desidrogenase (NADP)/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Mutação de Sentido Incorreto/genética , Sequência de Aminoácidos , Criança , Creatinina/urina , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Ligação Proteica/genética , Ligação Proteica/fisiologia , Homologia de Sequência de Aminoácidos , Timina/urina , Uracila/análogos & derivados , Uracila/urinaRESUMO
The reductive pyrimidine catabolic pathway is absent in Escherichia coli. However, the bacterium contains an enzyme homologous to mammalian dihydropyrimidine dehydrogenase. Here, we show that E. coli dihydropyrimidine dehydrogenase is the first member of a novel NADH-dependent subclass of iron-sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Proteínas de Escherichia coli/metabolismo , NAD/metabolismo , Uracila/análogos & derivados , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/genética , Dimerização , Escherichia coli/química , Escherichia coli/classificação , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Filogenia , Uracila/metabolismoRESUMO
In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.
Assuntos
Domínio Catalítico/genética , Di-Hidrouracila Desidrogenase (NADP)/genética , Mutagênese Sítio-Dirigida/métodos , Mutação , Substituição de Aminoácidos , Animais , Arginina/química , Arginina/genética , Arginina/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Glutamina/química , Glutamina/genética , Glutamina/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Cinética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Serina/química , Serina/genética , Serina/metabolismo , Espectrofotometria , SuínosRESUMO
A patient with hematuria was shown to have thymine-uraciluria. The dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells was 0.16 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h. Analysis of DPYD showed that the patient was compound heterozygous for the novel mutations 237C > A (C79X) in exon 4 and 704G > A (R235Q) in exon 7. The nonsense mutation (C79X) leads to premature termination of translation and thus to a non-functional protein. Analysis of the crystal structure of pig DPD suggested that the R235Q mutation might interfere with the binding of FAD and the electron flow between the NADPH and the pyrimidine substrate site of DPD.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/genética , Hematúria/enzimologia , Hematúria/genética , Mutação de Sentido Incorreto , Mutação Puntual , Adulto , Animais , Criança , Cristalografia por Raios X , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Feminino , Genótipo , Hematúria/patologia , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , SuínosRESUMO
5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Fluoruracila/farmacologia , Timidilato Sintase/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Deficiência da Di-Hidropirimidina Desidrogenase , Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Fenótipo , Interferência de RNA/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Timidilato Sintase/química , Timidilato Sintase/deficiência , Timidilato Sintase/genéticaRESUMO
Dihydropyrimidine dehydrogenase (DPD) is an inactivating and rate-limiting enzyme for 5-fluorouracil (5-FU), and its deficiency is associated with a risk for developing a severe or fatal toxicity to 5-FU. In this study, to search for genetic variations of DPYD encoding DPD in Japanese, the putative promoter region, all exons, and flanking introns of DPYD were sequenced from 341 subjects including cancer patients treated with 5-FU. Fifty-five genetic variations, including 38 novel ones, were found and consisted of 4 in the 5'-flanking region, 21 (5 synonymous and 16 nonsynonymous) in the coding exons, and 30 in the introns. Nine novel nonsynonymous SNPs, 29C>A (Ala10Glu), 325T>A (Tyr109Asn), 451A>G (Asn151Asp), 733A>T (Ile245Phe), 793G>A (Glu265Lys), 1543G>A (Val515Ile), 1572T>G (Phe524Leu), 1666A>C (Ser556Arg), and 2678A>G (Asn893Ser), were found at allele frequencies between 0.15 and 0.88%. Two known nonsynonymous variations reported only in Japanese, 1003G>T (*11, Val335Leu) and 2303C>A (Thr768Lys), were found at allele frequencies of 0.15 and 2.8%, respectively. SNP and haplotype distributions in Japanese were quite different from those reported previously in Caucasians. This study provides fundamental information for pharmacogenetic studies for evaluating the efficacy and toxicity of 5-FU in Japanese and probably East Asians.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Di-Hidrouracila Desidrogenase (NADP)/genética , Fluoruracila/toxicidade , Variação Genética , Povo Asiático/etnologia , Povo Asiático/genética , Di-Hidrouracila Desidrogenase (NADP)/química , Feminino , Frequência do Gene , Haplótipos , Humanos , Japão/etnologia , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A physiologically based pharmacokinetic (PBPK) model to simulate the plasma concentration and 13CO2 exhalation after [2-13C]uracil administration to DPD-suppressed dogs was developed. Simulation using this PBPK model should be useful in clinical situations where DPD-deficient patients at risk are to be detected with [2-13C]uracil as an in vivo probe.
Assuntos
Isótopos de Carbono/metabolismo , Deficiência da Di-Hidropirimidina Desidrogenase , Di-Hidrouracila Desidrogenase (NADP)/biossíntese , Uracila/sangue , Uracila/química , Uracila/farmacocinética , Administração Oral , Animais , Testes Respiratórios , Dióxido de Carbono/química , Simulação por Computador , Di-Hidrouracila Desidrogenase (NADP)/química , Cães , Cinética , Modelos Biológicos , Modelos Químicos , Modelos Teóricos , Fatores de TempoRESUMO
Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases, uracil and thymine, and is also known to be the key enzyme catalyzing the metabolic degradation of the anti-cancer drug 5-fluorouracil (5-FU). 5-FU has been commonly and widely used as a chemotherapeutic agent for the treatment of cancer of the gastrointestinal tract, breast, and head and neck. More than 85% of the administered 5-FU is catabolized by DPD. The clinical importance of DPD has been demonstrated with the identification of severe or lethal toxicity in patients administered 5-FU who are deficient in or have low levels of DPD activity in their peripheral blood mononuclear cells (PBMC). The importance of the role of DPD in 5-FU chemotherapy also has been shown by studies with competitive and irreversible DPD inhibitors. Population studies of DPD activity in PBMC were reported in healthy volunteers and cancer patients to evaluate the incidence of complete or partial DPD deficiency. In these studies, considerable variation was observed, and the frequency of low or deficient DPD activity (<30% and <10% of the mean activity of the normal population, respectively), was estimated to be 3-5% and 0.1%,respectively. We also found one healthy volunteer (0.7% of the population) with very low PBMC-DPD activity due to heterozygosity for a mutant allele of the DPYD gene in a population of 150 healthy Japanese volunteers. To date, at least 34 DPYD variants have been reported. However, genotyping of cancer patients with reduced or normal DPD activity showed that only 17% of those patients had a molecular basis for their deficient phenotype, which emphasized the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo,suggesting that it is difficult to identify DPD deficiency by genotyping. Therefore, it is important to develop methods for identifying DPD deficiency in cancer patients by phenotyping before 5-FU treatment.