RESUMO
AIMS: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. METHODS AND RESULTS: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. CONCLUSIONS: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.
Assuntos
Quimiotaxia de Leucócito , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Insuficiência Cardíaca/enzimologia , Leucócitos/metabolismo , Infarto do Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Função Ventricular Esquerda , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Quinase 5 de Receptor Acoplado a Proteína G/genética , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Camundongos Knockout , Contração Miocárdica , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Transdução de Sinais , Volume Sistólico , Transcriptoma , Pressão VentricularRESUMO
When myocardial function is compromised as in heart failure (HF), there is activation of the sympathetic nervous system with elevated circulating catecholamine levels. These catecholamines activate cardiac and extra-cardiac adrenergic receptors (ARs). Interest in secreted extracellular vesicles (EVs) from the heart is growing and in HF, it is not known whether excessive activation of α- or ß-adrenergic receptors (ARs) could induce specific changes in EV content. In this study, we have evaluated, by next generation sequencing, the small RNA content, including micro-RNAs (miRs), of circulating EVs of mice exposed to chronic selective α- or ß- AR stimulation. EVs from mouse blood were purified by differential ultracentrifugation resulting in EVs with an average size of 116.6 ± 4.8 nm that by immunoblotting included protein markers of EVs. We identified the presence of miRs in blood EVs using miR-21-5p and -16-5p real-time PCR as known constituents of blood exosomes that make up a portion of EVs. We next performed next generation sequencing (NGS) of small non-coding RNAs found in blood EVs from mice following 7 days of chronic treatment with isoproterenol (ISO) or phenylephrine (PE) to stimulate α- or ß-ARs, respectively. PE increased the percent of genomic repeat region reads and decreased the percent of miR reads. In miR expression analysis, PE and ISO displayed specific patterns of miR expression that suggests differential pathway regulation. The top 20 KEGG pathways predicted by differential expressed miRs show that PE and ISO share 11 of 20 pathways analyzed and reveal also key differences including three synapse relative pathways induced by ISO relative to PE treatment. Both α-and ß-AR agonists can alter small RNA content of circulating blood EVs/exosomes including differential expression and loading of miRs that indicate regulation of distinct pathways. This study provides novel insight into chronic sympathetic nervous system activation in HF where excessive catecholamines may not only participate in pathological remodeling of the heart but alter other organs due to secretion of EVs with altered miR content.
Assuntos
Doenças Cardiovasculares/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/sangue , Receptores Adrenérgicos alfa/sangue , Receptores Adrenérgicos beta/sangue , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , CamundongosRESUMO
Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase 5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca2+-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.
Assuntos
Cardiomegalia , Quinase 5 de Receptor Acoplado a Proteína G/genética , Insuficiência Cardíaca , Animais , Calmodulina/metabolismo , Cardiomegalia/genética , Núcleo Celular/metabolismo , Insuficiência Cardíaca/genética , Camundongos , Miócitos Cardíacos/metabolismoRESUMO
Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP-10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP-9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP-10 expression in UMSCC12 cells (p = 0.0001), and MMP-3 (p = 0.0005) and -9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP-2 (p = 0.0408) and MMP-9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP-2 (p = 0.0023) and -9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Movimento Celular , Neoplasias de Cabeça e Pescoço/enzimologia , Metaloproteinases da Matriz Secretadas/metabolismo , Glicoproteínas de Membrana/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metaloproteinases da Matriz Secretadas/genética , Glicoproteínas de Membrana/genética , Invasividade Neoplásica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço , TransfecçãoRESUMO
The enzyme isopenicillin N synthase (IPNS) installs the ß-lactam and thiazolidine rings of the penicillin core into the linear tripeptide l-δ-aminoadipoyl-l-Cys-d-Val (ACV) on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the ß-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C-H bond of the substrate: the pro-S-CCys,ß-H bond for closure of the ß-lactam ring, and the CVal,ß-H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as cosubstrate to cleave these C-H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C-H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C-H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin's founding work.
Assuntos
Aspergillus nidulans/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Cinética , Oxigênio/metabolismo , Teoria Quântica , Análise EspectralRESUMO
Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the ß1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Neoplasias de Cabeça e Pescoço/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Integrina beta1/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Invasividade Neoplásica , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Tempo , TransfecçãoRESUMO
The type I phosphoribosyltransferase OMP synthase (EC 2.4.2.10) is involved in de novo synthesis of pyrimidine nucleotides forming the UMP precursor orotidine 5'-monophosphate (OMP). The homodimeric enzyme has a Rossman α/ß core topped by a base-enclosing "hood" domain and a flexible domain-swapped catalytic loop. High-resolution X-ray structures of the homologous Salmonella typhimurium and yeast enzymes show that a general compacting of the core as well as movement of the hood and a major disorder-to-order transition of the loop occur upon binding of ligands MgPRPP and orotate. Here we present backbone NMR assignments for the unliganded yeast enzyme (49 kDa) and its complex with product OMP. We were able to assign 212-213 of the 225 non-proline backbone (15)N and amide proton resonances. Significant difference in chemical shifts of the amide cross peaks occur in regions of the structure that undergo movement upon ligand occupancy in the S. typhimurium enzyme.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Orotato Fosforribosiltransferase/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Biocatálise , Isótopos de Carbono , Hidrogênio , Ligantes , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/química , Uridina Monofosfato/metabolismoRESUMO
The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.
Assuntos
Domínio Catalítico , Orotato Fosforribosiltransferase/química , Orotato Fosforribosiltransferase/metabolismo , Saccharomyces cerevisiae/enzimologia , Escherichia coli/genética , Cinética , Modelos Moleculares , Mutação , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/isolamento & purificação , Fosforribosil Pirofosfato/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/metabolismoRESUMO
The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent enzymes are a functionally and mechanistically diverse group of mononuclear nonheme-iron enzymes that activate dioxygen to couple the decarboxylation of alphaKG, which yields succinate and CO(2), to the oxidation of an aliphatic C-H bond of their substrates. Their mechanisms have been studied in detail by a combination of kinetic, spectroscopic, and computational methods. Two reaction intermediates have been trapped and characterized for several members of this enzyme family. The first intermediate is the C-H-cleaving Fe(IV)-oxo complex, which exhibits a large deuterium kinetic isotope effect on its decay. The second intermediate is a Fe(II):product complex. Reaction intermediates proposed to occur before the Fe(IV)-oxo intermediate do not accumulate and therefore cannot be characterized experimentally. One of these intermediates is the initial O(2) adduct, which is a {FeO(2)}(8) species in the notation introduced by Enemark and Feltham. Here, we report spectroscopic and computational studies on the stable NO-adduct of taurine:alphaKG dioxygenase (TauD), termed TauD-{FeNO}(7), and its one-electron reduced form, TauD-{FeNO}(8). The latter is isoelectronic with the proposed O(2) adduct and was generated by low-temperature gamma-irradiation of TauD-{FeNO}(7). To our knowledge, TauD-{FeNO}(8) is the first paramagnetic {FeNO}(8) complex. The detailed analysis of experimental and computational results shows that TauD-{FeNO}(8) has a triplet ground state. This has mechanistic implications that are discussed in this Article. Annealing of the triplet {FeNO}(8) species presumably leads to an equally elusive {FeHNO}(8) complex with a quintet ground state.
Assuntos
Compostos Férricos/química , Compostos Ferrosos/química , Ferro/química , Oxigenases de Função Mista/química , Óxido Nítrico/química , Dióxido de Carbono/síntese química , Dióxido de Carbono/química , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/síntese química , Compostos Ferrosos/síntese química , Cinética , Magnetismo , Oxigenases de Função Mista/metabolismo , Conformação Molecular , Oxirredução , Espectroscopia de Mossbauer , Estereoisomerismo , Ácido Succínico/síntese química , Ácido Succínico/químicaRESUMO
The amine oxygenase AurF from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate (pABA) to p-nitrobenzoate (pNBA). In this work, we have studied the reaction of its reduced Fe(2)(II/II) cofactor with O(2), which results in generation of a peroxo-Fe(2)(III/III) intermediate. In the absence of substrate, this intermediate is unusually stable (t(1/2) = 7 min at 20 degrees C), allowing for its accumulation to almost stoichiometric amounts. Its decay is accelerated approximately 10(5)-fold by the substrate, pABA, implying that it is the complex that effects the two-electron oxidation of the amine to the hydroxylamine. The nearly quantitative conversion of pABA to pNBA by solutions containing an excess of the intermediate suggests that it may also be competent for the two subsequent two-electron oxidations leading to the product.
Assuntos
Ácido 4-Aminobenzoico/química , Compostos Férricos/química , Nitrobenzoatos/química , Oxigenases/química , Streptomyces/enzimologia , Catálise , Estabilidade Enzimática , OxirreduçãoRESUMO
PvdA catalyzes the hydroxylation of the side chain primary amine of ornithine in the initial step of the biosynthesis of the Pseudomonas aeruginosa siderophore pyoverdin. The reaction requires FAD, NADPH, and O(2). PvdA uses the same cosubstrates as several flavin-dependent hydroxylases that differ one from another in the kinetic mechanisms of their oxidative and reductive half-reactions. Therefore, the mechanism of PvdA was determined by absorption stopped-flow experiments. By contrast to some flavin-dependent hydroxylases (notably, p-hydroxybenzoate hydroxylase), binding of the hydroxylation target is not required to trigger reduction of the flavin by NADPH: the reductive half-reaction is equally facile in the presence and absence of ornithine. Reaction of O(2) with FADH(2) in the oxidative half-reaction is accelerated by ornithine 80-fold, providing a mechanism by which PvdA can ensure coupling of NADPH and ornithine oxidation. In the presence of ornithine, the expected C(4a)-hydroperoxyflavin intermediate with 390 nm absorption accumulates and decays to the C(4a)-hydroxyflavin in a kinetically competent fashion. The slower oxidative half-reaction that occurs in the absence of ornithine involves accumulation of an oxygenated flavin species and two subsequent states that are tentatively assigned as C(4a)-peroxy- and C(4a)-hydroperoxyflavin intermediates and the oxidized flavin. The enzyme generates stoichiometric hydrogen peroxide in lieu of hydroxyornithine. The data suggest that PvdA employs a kinetic mechanism that is a hybrid of those previously documented for other flavin-dependent hydroxylases.
Assuntos
Flavinas/química , Oxigenases de Função Mista/química , Oxigênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Catálise , Flavina-Adenina Dinucleotídeo/química , Peróxido de Hidrogênio/química , Hidrólise , Cinética , Oxigenases de Função Mista/metabolismo , Modelos Químicos , NADP/química , Ornitina/química , Oxigênio/química , Ligação Proteica , Especificidade por SubstratoRESUMO
Aliphatic halogenases activate O(2), cleave alpha-ketoglutarate (alphaKG) to CO(2) and succinate, and form haloferryl [X-Fe(IV)O; X = Cl or Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related alphaKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O(2) to form the C-H bond-cleaving ferryl complex is "triggered" by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-24-fold when the native l-Thr is replaced with another amino acid and by approximately 40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t(1/2) = 30-110 min at 0 degree C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
Assuntos
Carbono/química , Hidrogênio/química , Oxirredutases/química , Catálise , Cristalografia por Raios X , Cinética , Estrutura Molecular , Oxigênio/química , Oxigenases/química , Pseudomonas syringae/metabolismo , Espectrofotometria/métodos , Especificidade por Substrato , Temperatura , Fatores de TempoRESUMO
A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.
Assuntos
Chlamydia trachomatis/enzimologia , Compostos Ferrosos/metabolismo , Ferro/química , Manganês/química , Ribonucleotídeo Redutases/metabolismo , Sítios de Ligação , Catálise , Transporte de Elétrons , Compostos Ferrosos/química , Oxigênio/metabolismo , Conformação Proteica , Espectroscopia de MossbauerRESUMO
RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis.
Assuntos
Antivirais/farmacologia , Enterovirus Humano B/genética , Mutagênese/efeitos dos fármacos , Mutagênicos/farmacologia , Poliovirus/genética , Nucleosídeos de Purina/farmacologia , Enterovirus Humano B/fisiologia , Células HeLa/virologia , Humanos , Poliovirus/fisiologia , Nucleosídeos de Purina/químicaRESUMO
The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.
Assuntos
Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/metabolismo , Ferroproteínas não Heme/metabolismo , Oxigenases/metabolismo , Sítios de Ligação , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Catálise , Dicroísmo Circular , Halogenação , Histidina/química , Histidina/metabolismo , Ligação de Hidrogênio , Hidroxilação , Ácidos Cetoglutáricos/química , Ligantes , Oxigenases de Função Mista/química , Ferroproteínas não Heme/química , Oxigenases/química , Espectrofotometria Ultravioleta , TemperaturaAssuntos
Bromo/química , Grupo dos Citocromos c/química , Compostos Férricos/química , Ácidos Cetoglutáricos/química , Oxirredutases/química , Análise Espectral/métodos , Streptomyces/enzimologia , Catálise , Compostos Férricos/síntese química , Halogenação , Conformação Molecular , Oxirredução , Estereoisomerismo , Raios XRESUMO
In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.
Assuntos
Chlamydia trachomatis/enzimologia , Coenzimas/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Ribonucleotídeo Redutases/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Ativação Enzimática , Compostos Férricos/metabolismo , Tirosina/metabolismoRESUMO
The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases activate O2 for cleavage of unactivated C-H bonds in their substrates. The key intermediate that abstracts hydrogen in the reaction of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, was recently characterized. The intermediate, denoted J, was shown to contain an iron(IV)-oxo unit. Other important structural features of J, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere, are not yet understood. To probe these important structural features, a series of models for J with the Fe(IV) ion coordinated by the expected two imidazole (from His99 and His255), two carboxylate (succinate and Asp101), and oxo ligands have been generated by density functional theory (DFT) calculations, and spectroscopic parameters (Mössbauer isomer shift, quadrupole splitting, and asymmetry parameter, 57Fe hyperfine coupling tensor, and zero field splitting parameters, D and E/D) have been calculated for each model. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data. Similar analysis of the Fe(IV) complex generated in the variant protein with His99, the residue that contributes the imidazole ligand cis to the oxo group, replaced by alanine suggests that the deleted imidazole is replaced by a water ligand. This work lends credence to the idea that the combination of Mössbauer spectroscopy and DFT calculations can provide detailed structural information for reactive intermediates in the catalytic cycles of iron enzymes.
Assuntos
Biologia Computacional/métodos , Escherichia coli/enzimologia , Ferro/química , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Ferroproteínas não Heme/química , Alanina/genética , Substituição de Aminoácidos , Histidina/genética , Ligação de Hidrogênio , Ligantes , Modelos Químicos , Oxigênio/química , Espectroscopia de MossbauerRESUMO
Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products. Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require alpha-ketoglutarate (alphaKG, 1), chloride and oxygen. To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the gamma-methyl substituent of L-2-aminobutyric acid (L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase (CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe(IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe(IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and alphaKG-dependent hydroxylases.