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1.
Nucleic Acids Res ; 50(7): 4148-4160, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35333330

RESUMO

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , RNA , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/química , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Desmetilação , Humanos , RNA/química
2.
Proteins ; 91(11): 1510-1524, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37449559

RESUMO

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases , Isoformas de Proteínas
3.
Proc Natl Acad Sci U S A ; 117(37): 23140-23147, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868422

RESUMO

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oxigênio/metabolismo , Cisteína Dioxigenase/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Oxirredução , Transdução de Sinais/fisiologia , Fatores de Transcrição
4.
Angew Chem Int Ed Engl ; 61(45): e202211510, 2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36112310

RESUMO

Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high-throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl-hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone-based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.


Assuntos
Descoberta de Drogas , Prolina Dioxigenases do Fator Induzível por Hipóxia , Humanos , Polarização de Fluorescência , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo
5.
J Biol Chem ; 295(49): 16545-16561, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-32934009

RESUMO

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.


Assuntos
Dictyostelium/enzimologia , Prolil Hidroxilases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cinética , Simulação de Dinâmica Molecular , Oxigênio/metabolismo , Prolil Hidroxilases/química , Prolil Hidroxilases/genética , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
6.
Bioinformatics ; 36(3): 904-909, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504189

RESUMO

MOTIVATION: Metalloenzymes are attractive targets for therapeutic intervention owing to their central roles in various biological processes and pathological situations. The fast-growing body of structural data on metalloenzyme-ligand interactions is facilitating efficient drug discovery targeting metalloenzymes. However, there remains a shortage of specific databases that can provide centralized, interconnected information exclusive to metalloenzyme-ligand associations. RESULTS: We created a Metalloenzyme-Ligand Association Database (MeLAD), which is designed to provide curated structural data and information exclusive to metalloenzyme-ligand interactions, and more uniquely, present expanded associations that are represented by metal-binding pharmacophores (MBPs), metalloenzyme structural similarity (MeSIM) and ligand chemical similarity (LigSIM). MeLAD currently contains 6086 structurally resolved interactions of 1416 metalloenzymes with 3564 ligands, of which classical metal-binding, non-classical metal-binding, non-metal-binding and metal water-bridging interactions account for 63.0%, 2.3%, 34.4% and 0.3%, respectively. A total of 263 monodentate, 191 bidentate and 15 tridentate MBP chemotypes were included in MeLAD, which are linked to different active site metal ions and coordination modes. 3726 and 52 740 deductive metalloenzyme-ligand associations by MeSIM and LigSIM analyses, respectively, were included in MeLAD. An online server is provided for users to conduct metalloenzyme profiling prediction for small molecules of interest. MeLAD is searchable by multiple criteria, e.g. metalloenzyme name, ligand identifier, functional class, bioinorganic class, metal ion and metal-containing cofactor, which will serve as a valuable, integrative data source to foster metalloenzyme related research, particularly involved in drug discovery targeting metalloenzymes. AVAILABILITY AND IMPLEMENTATION: MeLAD is accessible at https://melad.ddtmlab.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Metaloproteínas , Domínio Catalítico , Descoberta de Drogas , Ligantes , Metais
7.
Angew Chem Int Ed Engl ; 60(26): 14657-14663, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33887099

RESUMO

Aspartate/asparagine-ß-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.


Assuntos
Compostos Ferrosos/metabolismo , Oxigenases de Função Mista/metabolismo , Asparagina/química , Asparagina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Biocatálise , Cristalografia por Raios X , Compostos Ferrosos/química , Humanos , Ligantes , Oxigenases de Função Mista/genética , Modelos Moleculares
8.
J Biol Chem ; 294(30): 11637-11652, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31147442

RESUMO

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Catálise , Cristalografia por Raios X , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Humanos , Hidroxilação , Histona Desmetilases com o Domínio Jumonji/química , Cinética , Lisina/metabolismo , Conformação Proteica , Especificidade por Substrato
9.
Nature ; 510(7505): 422-426, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24814345

RESUMO

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.


Assuntos
Eucariotos/enzimologia , Modelos Moleculares , Oxigenases/química , Células Procarióticas/enzimologia , Ribossomos/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Eucariotos/classificação , Humanos , Oxigenases/metabolismo , Filogenia , Células Procarióticas/classificação , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência
10.
Proc Natl Acad Sci U S A ; 114(18): 4667-4672, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28420789

RESUMO

Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the four-electron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of l-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.


Assuntos
Proteínas de Bactérias/química , Etilenos/química , Ácidos Cetoglutáricos/química , Liases/química , Modelos Químicos , Pseudomonas syringae/enzimologia , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Etilenos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Liases/metabolismo
11.
Extremophiles ; 22(3): 553-562, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29523972

RESUMO

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960-962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective.


Assuntos
Proteínas de Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Rhodothermus/enzimologia , Proteínas Ribossômicas/metabolismo , Estabilidade Enzimática , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroxilação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Homologia de Sequência
12.
Bioorg Med Chem ; 26(11): 2928-2936, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29655609

RESUMO

Metallo-ß-lactamases (MBLs) enable bacterial resistance to almost all classes of ß-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging 'hydrolytic' water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.


Assuntos
Rodanina/química , Compostos de Sulfidrila/química , Inibidores de beta-Lactamases/síntese química , beta-Lactamases/química , Enedi-Inos/química , Concentração Inibidora 50 , Estrutura Molecular , Rodanina/síntese química , Rodanina/farmacologia , Relação Estrutura-Atividade , Compostos de Sulfidrila/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos
13.
Hum Mol Genet ; 24(9): 2458-69, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25596185

RESUMO

The ethylmalonic encephalopathy protein 1 (ETHE1) catalyses the oxygen-dependent oxidation of glutathione persulfide (GSSH) to give persulfite and glutathione. Mutations to the hETHE1 gene compromise sulfide metabolism leading to the genetic disease ethylmalonic encephalopathy. hETHE1 is a mono-iron binding member of the metallo-ß-lactamase (MBL) fold superfamily. We report crystallographic analysis of hETHE1 in complex with iron to 2.6 Å resolution. hETHE1 contains an αßßα MBL-fold, which supports metal-binding by the side chains of an aspartate and two histidine residues; three water molecules complete octahedral coordination of the iron. The iron binding hETHE1 enzyme is related to the 'classical' di-zinc binding MBL hydrolases involved in antibiotic resistance, but has distinctive features. The histidine and aspartate residues involved in iron-binding in ETHE1, occupy similar positions to those observed across both the zinc 1 and zinc 2 binding sites in classical MBLs. The active site of hETHE1 is very similar to an ETHE1-like enzyme from Arabidopsis thaliana (60% sequence identity). A channel leading to the active site is sufficiently large to accommodate a GSSH substrate. Some of the observed hETHE1 clinical mutations cluster in the active site region. The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors.


Assuntos
Proteínas Mitocondriais/química , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Domínio Catalítico , Ativação Enzimática , Humanos , Metais/química , Metais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Púrpura/genética , Púrpura/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
14.
Artigo em Inglês | MEDLINE | ID: mdl-28115348

RESUMO

ß-Lactamase-mediated resistance is a growing threat to the continued use of ß-lactam antibiotics. The use of the ß-lactam-based serine-ß-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-ß-lactam inhibitor avibactam has extended the utility of ß-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-ß-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-ß-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of "dual-action" inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-ß-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of ß-lactamase, including the class A extended spectrum ß-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of ß-lactams against Gram-negative clinical isolates expressing a variety of ß-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other ß-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-ß-lactamase catalysis.


Assuntos
Antibacterianos/farmacologia , Ácidos Borônicos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Motivos de Aminoácidos , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ácidos Borônicos/síntese química , Clonagem Molecular , Cristalografia por Raios X , Ciclização , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Resistência beta-Lactâmica/genética , Inibidores de beta-Lactamases/síntese química , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
15.
Org Biomol Chem ; 15(28): 6024-6032, 2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28678295

RESUMO

The class D (OXA) serine ß-lactamases are a major cause of resistance to ß-lactam antibiotics. The class D enzymes are unique amongst ß-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that ß-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D ß-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.


Assuntos
Compostos Azabicíclicos/farmacologia , Halogênios/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Compostos Azabicíclicos/química , Isótopos de Carbono , Cristalografia por Raios X , Halogênios/química , Íons/química , Íons/farmacologia , Modelos Moleculares , Conformação Molecular , Inibidores de beta-Lactamases/química
16.
J Med Genet ; 53(3): 200-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26378117

RESUMO

BACKGROUND: A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. METHODS: We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. RESULTS: We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity. CONCLUSION: Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.


Assuntos
Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto , Proteínas/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente
17.
Proc Natl Acad Sci U S A ; 111(11): 4019-24, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24550462

RESUMO

The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations.


Assuntos
Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Ribossômicas/metabolismo , Ribossomos/fisiologia , Clorófitas , Códon de Terminação/genética , Humanos , Hidroxilação , Espectrometria de Massas , Oxigenases/genética , Oxigenases/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces , Especificidade da Espécie
18.
Proc Natl Acad Sci U S A ; 111(37): 13331-6, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25197067

RESUMO

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.


Assuntos
Oxigênio/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Prolina/metabolismo , Pseudomonas putida/metabolismo , Chlamydomonas reinhardtii/metabolismo , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fator Tu de Elongação de Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
19.
Antimicrob Agents Chemother ; 60(1): 142-50, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26482303

RESUMO

ß-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-ß-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all ß-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-ß-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Inibidores da Enzima Conversora de Angiotensina/química , Antibacterianos/farmacologia , Captopril/química , Carbapenêmicos/farmacologia , Clonagem Molecular , Cristalografia por Raios X , Reposicionamento de Medicamentos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Expressão Gênica , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , Inibidores de beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
20.
Chemistry ; 22(4): 1270-6, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26660433

RESUMO

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe(II) - and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of γ-butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N(+) >P(+) >As(+). The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-π interactions in productive substrate recognition.


Assuntos
Betaína/análogos & derivados , Carnitina/química , Cátions/química , Compostos de Amônio Quaternário/química , gama-Butirobetaína Dioxigenase/química , Betaína/química , Catálise , Cinética , Oxirredução , Ligação Proteica , gama-Butirobetaína Dioxigenase/metabolismo
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