RESUMEN
AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de la Membrana/química , Oxigenasas de Función Mixta/química , Proteínas Musculares/química , Secuencia de Aminoácidos , Asparagina/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Dominio Catalítico , Cristalografía , Disulfuros/química , Disulfuros/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Conformación ProteicaRESUMEN
Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.
Asunto(s)
Aldehídos/metabolismo , Ésteres/metabolismo , Oxidorreductasas/metabolismo , Compuestos de Sulfhidrilo/química , Aldehídos/química , Sitios de Unión , Biocatálisis , Dominio Catalítico , Cefalosporinas/biosíntesis , Cefalosporinas/química , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Ésteres/química , Hierro/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Molecular , Mutagénesis , Oxidación-Reducción , Oxidorreductasas/genética , Oxígeno/química , Oxígeno/metabolismo , Penicilinas/biosíntesis , Penicilinas/química , Especificidad por SustratoRESUMEN
Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines.
Asunto(s)
Hidrazinas/química , Hidrazinas/farmacología , Hierro/metabolismo , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Simulación del Acoplamiento Molecular , Procolágeno-Prolina Dioxigenasa/química , Unión Proteica/efectos de los fármacos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Aromatic analogues of the 2-oxoglutarate co-substrate of the hypoxia-inducible factor hydroxylases are shown to bind at the active site iron: Pyridine-2,4-dicarboxylate binds as anticipated with a single molecule chelating the iron in a bidentate manner. The binding mode of a hydroxamic acid analogue, at least in the crystalline state, is unusual because two molecules of the inhibitor are observed at the active site and partial displacement of the iron binding aspartyl residue was observed.
Asunto(s)
Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/farmacología , Proteínas Represoras/metabolismo , Sitios de Unión , Dominio Catalítico , Humanos , Oxigenasas de Función Mixta , Modelos Moleculares , Unión Proteica , Proteínas Represoras/químicaRESUMEN
How PHDs achieve specificity: trans-4-prolyl hydroxylation of the transcription factor HIF occurs with stereochemical retention. Substrate-analogue studies show how the von Hippel Lindau tumor suppressor protein (pVHL) and the oxygen-sensing hydroxylases (PHDs) achieve specificity for hydroxyprolyl/prolyl residues for the C(4)-exo/endo prolyl conformations, respectively.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Técnicas Biosensibles , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.
Asunto(s)
ADN/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Núcleo Celular/enzimología , Biología Computacional , Metilación de ADN , ADN de Cadena Simple/metabolismo , Ingestión de Alimentos , Metabolismo Energético , Ayuno , Compuestos Ferrosos/metabolismo , Hipotálamo/enzimología , Hipotálamo/metabolismo , Masculino , Ratones , Oxigenasas de Función Mixta , Datos de Secuencia Molecular , Oxo-Ácido-Liasas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Ácido Succínico/metabolismo , Timina/análogos & derivados , Timina/metabolismoRESUMEN
In humans both the levels and activity of the alpha-subunit of the hypoxia-inducible transcription factor (HIF-alpha) are regulated by its post-translation hydroxylation as catalyzed by iron- and 2-oxoglutarate (2OG)-dependent prolyl and asparaginyl hydroxylases (PHD1-3 and factor-inhibiting HIF (FIH), respectively). One consequence of hypoxia is the accumulation of tricarboxylic acid cycle intermediates (TCAIs). In vitro assays were used to assess non-2OG TCAIs as inhibitors of purified PHD2 and FIH. Under the assay conditions, no significant FIH inhibition was observed by the TCAIs or pyruvate, but fumarate, succinate, and isocitrate inhibited PHD2. Mass spectrometric analyses under nondenaturing conditions were used to investigate the binding of TCAIs to PHD2 and supported the solution studies. X-ray crystal structures of FIH in complex with Fe(II) and fumarate or succinate revealed similar binding modes for each in the 2OG co-substrate binding site. The in vitro results suggest that the cellular inhibition of PHD2, but probably not FIH, by fumarate and succinate may play a role in the Warburg effect providing that appropriate relative concentrations of the components are achieved under physiological conditions.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo del Ácido Cítrico , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Oxigenasas de Función Mixta/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Mama/enzimología , Mama/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Lactógeno Placentario/metabolismo , Proteína-Lisina 6-Oxidasa/genéticaRESUMEN
Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-alpha subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 A resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded beta-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.
Asunto(s)
Dominio Catalítico , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/genética , Conformación Proteica , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
Cyclic beta-oxocarboxylic acids inhibit factor inhibiting hypoxia-inducible factor via ligation to the active site iron.
Asunto(s)
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Proteínas Represoras/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Modelos Moleculares , Oxigenasas/metabolismo , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The ferrous iron and 2-oxoglutarate (2OG) dependent oxygenases catalyse two electron oxidation reactions by coupling the oxidation of substrate to the oxidative decarboxylation of 2OG, giving succinate and carbon dioxide coproducts. The evidence available on the level of incorporation of one atom from dioxygen into succinate is inconclusive. Here, we demonstrate that five members of the 2OG oxygenase family, AlkB from Escherichia coli, anthocyanidin synthase and flavonol synthase from Arabidopsis thaliana, and prolyl hydroxylase domain enzyme 2 and factor inhibiting hypoxia-inducible factor-1 from Homo sapiens all incorporate a single oxygen atom, almost exclusively derived from dioxygen, into the succinate co-product.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxígeno/metabolismo , Oxigenasas/metabolismo , Proteínas de Plantas/metabolismo , Ácido Succínico/metabolismo , Proteínas Bacterianas/química , Humanos , Hierro/química , Ácidos Cetoglutáricos/química , Estructura Molecular , Oxidación-Reducción , Oxígeno/química , Isótopos de Oxígeno/química , Isótopos de Oxígeno/metabolismo , Oxigenasas/química , Proteínas de Plantas/química , Ácido Succínico/químicaRESUMEN
A set of four non-heme iron(II) and 2-oxoglutarate-dependent enzymes catalyze the post-translational modification of a transcription factor, hypoxia inducible factor (HIF), that mediates the hypoxic response in animals. Hydroxylation of HIF both causes its degradation and limits its activity. We describe how the use of structural data coupled to solid-phase synthesis led to the discovery of a selective inhibitor of one of the HIF hydroxylases. The inhibitor N-oxalyl-d-phenylalanine was shown to inhibit the HIF asparaginyl hydroxylase (FIH) but not a HIF prolyl hydroxylase. A crystal structure of the inhibitor complexed to FIH reveals that it binds in the 2OG and, likely, in the dioxygen binding site. The results will help to enable the modulation of the hypoxic response for the up-regulation of specific genes of biomedical importance, such as erythropoietin and vascular endothelial growth factor.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Cinética , Oxigenasas de Función Mixta , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Estructura Secundaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Chlorination-elimination chemistry coupled with three-component Joullié-Ugi reaction and facile deprotection allowed efficient access to an array of polyhydroxylated pyrrolidines through parallel synthesis that may be considered to be a library of imino (aza) sugars (glycomimetics) and/or dihydroxyprolyl peptides (peptidomimetics). The utility of generating such a library was illustrated by screening against 15 different targets that revealed potent and selective inhibition of the Gaucher's disease glycosyltransferase enzyme glucosylceramide synthase and of primary pathogen model for human hepatitis C virus (HCV) and bovine diarrhoeal virus (BVDV). An observed selectivity for this HCV model over hepatitis B virus and remarkably low toxicity suggest a novel mode of action.
Asunto(s)
Antivirales/química , Materiales Biomiméticos/química , Glicopéptidos/química , Pirrolidinas/química , Antivirales/farmacología , Compuestos Aza/química , Compuestos Aza/farmacología , Materiales Biomiméticos/farmacología , Carbohidratos/química , Carbohidratos/farmacología , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Eritritol/química , Eritritol/farmacología , Glicopéptidos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Virus de la Hepatitis B/efectos de los fármacos , Hidroxiprolina/análogos & derivados , Hidroxiprolina/farmacología , Pirrolidinas/farmacología , Alcoholes del Azúcar/química , Alcoholes del Azúcar/farmacologíaRESUMEN
Regulation of the hypoxic response in humans is regulated by the post-translational hydroxylation of hypoxia inducible transcription factor; a recombinant form of a human prolyl-4-hydroxylase (PHD2) was characterised and shown to have an unexpectedly high affinity for, and to copurify with endogenous levels of, its Fe(ii) cofactor and 2-oxoglutarate cosubstrate.
Asunto(s)
Compuestos Ferrosos/química , Factor 1 Inducible por Hipoxia/química , Ácidos Cetoglutáricos/química , Procolágeno-Prolina Dioxigenasa/química , Sitios de Unión , Cromatografía Liquida , Humanos , Espectrometría de Masas , Modelos MolecularesRESUMEN
HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.
Asunto(s)
Factores de Transcripción/antagonistas & inhibidores , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Arginina/genética , Arginina/fisiología , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Dimerización , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Leucina/genética , Leucina/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/genética , Mutagénesis Sitio-Dirigida/fisiología , Mutación Missense/genética , Mutación Missense/fisiología , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Péptidos/fisiología , Fosforilación , Mutación Puntual/genética , Ratas , Espectrometría de Masa por Ionización de Electrospray/métodos , Especificidad por Sustrato/genética , Especificidad por Sustrato/fisiología , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteínas de Xenopus/química , Proteínas de Pez Cebra/químicaRESUMEN
Hydroxylation of hypoxia-inducible factor, a nuclear transcription factor, is catalysed by iron and 2-oxoglutarate dependent hydroxylases. Various analogues of the 2-oxoglutarate cosubstrate were synthesised and shown to inhibit the activity of human hypoxia-inducible factor-1alpha prolyl hydroxylases in cell-free extracts.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Ácidos Cetoglutáricos/síntesis química , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Catálisis , Sistema Libre de Células , Inhibidores Enzimáticos/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hierro/química , Quelantes del Hierro/farmacología , Isoenzimas/antagonistas & inhibidores , Ácidos Cetoglutáricos/farmacología , Estructura MolecularRESUMEN
Analogues of the naturally occurring cyclic hydroxamate dealanylalahopcin, which is an inhibitor of procollagen prolyl-4-hydroxylase, were synthesised and shown to be inhibitors of the human hypoxia-inducible factor prolyl hydroxylases.
Asunto(s)
Aminobutiratos/química , Aminobutiratos/farmacología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Aminobutiratos/síntesis química , Aminobutiratos/metabolismo , Animales , Autorradiografía , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hierro/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/metabolismoRESUMEN
AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans. Recent analyses have verified the prediction that AlkB is a member of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family of enzymes. AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytosine lesions. Other members of the Fe(II) and 2OG-dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention. Assays measuring 2OG turnover were used to investigate the selectivity of AlkB. 1-Methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, and 3-methyl-2'-deoxycytidine all stimulated 2OG turnover by AlkB but were not demethylated indicating an uncoupling of 2OG and prime substrate oxidation and that oligomeric DNA is required for hydroxylation and subsequent demethylation. In contrast the equivalent unmethylated nucleosides did not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG. Stimulation of 2OG turnover by 1-methyladenosine was highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol. Following the observation that AlkB is inhibited by high concentrations of 2OG, analogues of 2OG, including 2-mercaptoglutarate, were found to specifically inhibit AlkB. The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor-inhibiting hypoxia-inducible factor (FIH) in vitro. FIH inhibition by quercetin occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB by quercetin is, predominantly, due to nonspecific iron chelation.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Oxigenasas de Función Mixta/antagonistas & inhibidores , Ácido Ascórbico/farmacología , Proteínas de Escherichia coli/metabolismo , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Quercetina/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
The activity of the transcription factor hypoxia-inducible factor (HIF) is regulated by oxygen-dependent hydroxylation. Under normoxic conditions, hydroxylation of proline residues triggers destruction of its alpha-subunit while hydroxylation of Asn(803) in the C-terminal transactivation domain of HIF-1 alpha (CAD) prevents its interaction with p300. Here we report crystal structures of the asparagine hydroxylase (factor-inhibiting HIF, FIH) complexed with Fe((II)), 2-oxoglutarate cosubstrate, and CAD fragments, which reveal the structural basis of HIF modification. CAD binding to FIH occurs via an induced fit process at two distinct interaction sites. At the hydroxylation site CAD adopts a loop conformation, contrasting with a helical conformation for the same residues when bound to p300. Asn(803) of CAD is buried and precisely orientated in the active site such that hydroxylation occurs at its beta-carbon. Together with structures with the inhibitors Zn((II)) and N-oxaloylglycine, analysis of the FIH-CAD complexes will assist design of hydroxylase inhibitors with proangiogenic properties. Conserved structural motifs within FIH imply it is one of an extended family of Fe((II)) oxygenases involved in gene regulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Factor 1 Inducible por Hipoxia , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Oxidación-Reducción , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer.
Asunto(s)
Asparagina/metabolismo , Carbono/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Catálisis , Proteínas de Unión al ADN/química , Hidroxilación , Factor 1 Inducible por Hipoxia , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/químicaRESUMEN
The hypoxic response in animals is mediated by hydroxylation of proline residues in the alpha-subunit of hypoxia inducible factor (HIF). Hydroxylation is catalysed by prolyl-4-hydroxylases (PHD isozymes in humans) which are iron(II) and 2-oxoglutarate dependent oxygenases. Mutation of the arginine proposed to bind 2-oxoglutarate and of the 2His-1-carboxylate iron(II) binding motif in PHD1 dramatically reduces its activity. The source of the oxygen of the product alcohol is (>95%) dioxygen.