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
A ß-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Malassezia globosa has been cloned, characterized, and studied for its inhibition with sulfonamides. This enzyme, designated MG-CA, has significant catalytic activity in the CO(2) hydration reaction and was inhibited by sulfonamides, sulfamates, and sulfamides with K(I) in the nanomolar to micromolar range. Several sulfonamides have also been investigated for the inhibition of growth of M. globosa, M. dermatis, M. pachydermatic, and M. furfur in cultures, whereas a mouse model of dandruff showed that treatment with sulfonamides led to fragmented fungal hyphae, as for the treatment with ketoconazole, a clinically used antifungal agent. These data prompt us to propose MG-CA as a new antidandruff drug target.
Asunto(s)
Antifúngicos/química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Dermatomicosis/microbiología , Proteínas Fúngicas/química , Malassezia/enzimología , Dermatosis del Cuero Cabelludo/microbiología , Secuencia de Aminoácidos , Animales , Antifúngicos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/genética , Clonación Molecular , Dermatomicosis/tratamiento farmacológico , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/genética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Malassezia/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Dermatosis del Cuero Cabelludo/tratamiento farmacológico , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacologíaRESUMEN
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
(3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A beta-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2(1) space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the "second" CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins.
Asunto(s)
Acetiltransferasas/química , Ácido Clavulánico/biosíntesis , Redes y Vías Metabólicas , Espectrometría de Masa por Ionización de Electrospray , Streptomyces/enzimología , Acetilcoenzima A/metabolismo , Sitios de Unión , Ácido Clavulánico/química , Cristalografía por Rayos X , Modelos Moleculares , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The oxygen-dependent hydroxylation of proline residues in the alpha subunit of hypoxia-inducible transcription factor (HIFalpha) is central to the hypoxic response in animals. Prolyl hydroxylation of HIFalpha increases its binding to the von Hippel-Lindau protein (pVHL), so signaling for degradation via the ubiquitin-proteasome system. The HIF prolyl hydroxylases (PHDs, prolyl hydroxylase domain enzymes) are related to the collagen prolyl hydroxylases, but form unusually stable complexes with their Fe(II) cofactor and 2-oxoglutarate cosubstrate. We report crystal structures of the catalytic domain of PHD2, the most important of the human PHDs, in complex with the C-terminal oxygen-dependent degradation domain of HIF-1alpha. Together with biochemical analyses, the results reveal that PHD catalysis involves a mobile region that isolates the hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. The results will be of use in the design of PHD inhibitors aimed at treating anemia and ischemic disease.
Asunto(s)
Factor 1 Inducible por Hipoxia/química , Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Hidroxilación , Hidroxiprolina/metabolismo , Hipoxia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Prolina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.
Asunto(s)
Metilación de ADN , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxigenasas de Función Mixta/metabolismo , Secuencia de Bases , Biocatálisis , ADN/genética , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Concentración 50 Inhibidora , Oxigenasas de Función Mixta/antagonistas & inhibidoresRESUMEN
A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.
Asunto(s)
Histidina/química , Hierro/química , Proteínas Represoras/química , Catálisis , Cristalografía por Rayos X/métodos , Dioxigenasas/química , Hemo/química , Humanos , Ácidos Cetoglutáricos/química , Ligandos , Metales/química , Oxigenasas de Función Mixta , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Proteínas Represoras/metabolismoRESUMEN
Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta2 and beta3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta2beta3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta2beta3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Secuencia de Aminoácidos , Hidroxilación , Factor 1 Inducible por Hipoxia/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
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
The adaptation of animals to oxygen availability is mediated by a transcription factor termed hypoxia-inducible factor (HIF). HIF is an alpha (alpha)/beta (beta) heterodimer that binds hypoxia response elements (HREs) of target genes, including some of medicinal importance, such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). While the concentration of the HIF-beta subunit, a constitutive nuclear protein, does not vary with oxygen availability, the abundance and activity of the HIF-alpha subunits are tightly regulated via oxygen-dependent modification of specific residues. Hydroxylation of prolyl residues (Pro402 and Pro564 in HIF-1alpha) promotes interaction with the von Hippel-Lindau E3 ubiquitin ligase and, consequently, proteolytic destruction by the ubiquitin-proteasome pathway. This prolyl hydroxylation is catalyzed by the prolyl-hydroxylase domain (PHD) containing enzymes for which three isozymes have been identified in humans (1-3). Additionally, asparaginyl hydroxylation (Asn803 in HIF-1alpha) by factor-inhibiting HIF (FIH) ablates interaction of the HIF-alpha subunit with the coactivator p300, providing an alternative mechanism for down-regulation of HIF-dependent genes. Under hypoxic conditions, when oxygen-mediated regulation of the alpha-subunits is curtailed or minimized, dimerization of the alpha- and beta-subunits occurs with subsequent target gene upregulation. Therapeutic activation of HIF signaling has been suggested as a potential treatment for numerous conditions, including ischemia, stroke, heart attack, inflammation, and wounding. One possible route to achieve this is via inhibition of the HIF hydroxylases. This chapter details methods for the purification and assaying of PHD2, the most abundant PHD and the most important in setting steady-state levels of HIF-alpha. Assays are described that measure the activity of PHD2 via direct and indirect means. Furthermore, conditions for the screening of small molecules against PHD2 are described.
Asunto(s)
Factor 1 Inducible por Hipoxia/química , Procolágeno-Prolina Dioxigenasa/análisis , Procolágeno-Prolina Dioxigenasa/química , Animales , Dióxido de Carbono/química , Cromatografía Liquida , Escherichia coli/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Espectrometría de Masas , Oxígeno/química , Consumo de Oxígeno , Procolágeno-Prolina Dioxigenasa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Succinatos/análisis , Succinatos/químicaRESUMEN
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.
Asunto(s)
Repetición de Anquirina , Receptores Notch/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Asparagina/metabolismo , Cristalografía por Rayos X , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxigenasas de Función Mixta , Estructura Terciaria de Proteína , Receptor Notch1/metabolismo , Receptor Notch2 , Receptor Notch3 , Receptores Notch/química , Proteínas Represoras/química , Factores de Transcripción/químicaRESUMEN
The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.
Asunto(s)
Consumo de Oxígeno , Procolágeno-Prolina Dioxigenasa/metabolismo , Clonación Molecular , Glucosa Oxidasa/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Cinética , Oxigenasas de Función Mixta , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Especificidad por Sustrato , Factores de Transcripción/genética , Factores de Transcripción/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
Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IkappaB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IkappaBalpha were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.
Asunto(s)
Repetición de Anquirina , Factor 1 Inducible por Hipoxia/metabolismo , Proteínas I-kappa B/química , Proteínas I-kappa B/metabolismo , Oxigenasas de Función Mixta/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Descarboxilación , Humanos , Hidroxilación , Ácidos Cetoglutáricos/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Subunidad p50 de NF-kappa B/análisis , Subunidad p50 de NF-kappa B/química , Subunidad p50 de NF-kappa B/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Factores de Transcripción/químicaRESUMEN
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
In humans, many responses to hypoxia including angiogenesis and erythropoiesis are mediated by the alpha/beta-heterodimeric transcription factor hypoxia inducible factor (HIF). The stability and/or activity of human HIF-1alpha are modulated by post-translational modifications including prolyl and asparaginyl hydroxylation, phosphorylation, and reportedly by acetylation of the side-chain of Lys532 by ARD1 (arrest defective protein 1 homologue), an acetyltransferase. Using purified recombinant human ARD1 (hARD1) we did not observe ARD1-mediated N-acetylation of Lys532 using fragments of HIF-1alpha. However, recombinant hARD1 from Escherichia coli was produced with partial N-terminal acetylation and was observed to undergo slow self-mediated N-terminal acetylation. The observations are consistent with the other data indicating that hARD1, at least alone, does not acetylate HIF-1alpha, and with reports on the N-terminal acetyltransferase activity of a recently reported heterodimeric complex comprising hARD1 and N-acetyltransferase protein.
Asunto(s)
Acetiltransferasas/aislamiento & purificación , Acetiltransferasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lisina/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Acetilación , Acetiltransferasas/análisis , Acetiltransferasas/química , Hormona Adrenocorticotrópica/metabolismo , Catálisis , Cromatografía Liquida , Histidina/metabolismo , Humanos , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/análisis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
(3R,5R)-Clavulanic acid, a clinically used inhibitor of serine beta-lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic beta-lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis.
Asunto(s)
Ácido Clavulánico/biosíntesis , Glicina/metabolismo , Hidrolasas/genética , Hidrolasas/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Ácido Clavulánico/química , Codón Iniciador , Fermentación , Genes Bacterianos/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad por SustratoRESUMEN
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
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
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.