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1.
Science ; 368(6489): 424-427, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32217749

RESUMO

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.


Assuntos
Proteínas de Escherichia coli/química , Ribonucleotídeo Redutases/química , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Conformação Proteica , Ribonucleotídeo Redutases/genética , Tirosina/química
2.
Dalton Trans ; 49(6): 1742-1746, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31967142

RESUMO

The reactivity of the previously reported peroxo-adduct [FeIII2(µ-O)(µ-1,2-O2)(IndH)2(solv)2]2+ (1) (IndH = 1,3-bis(2-pyridyl-imino)isoindoline) has been investigated in nucleophilic (e.g., deformylation of alkyl and aryl alkyl aldehydes) and electrophilic (e.g. oxidation of phenols) stoichiometric reactions as biomimics of ribonucleotide reductase (RNR-R2) and aldehyde deformylating oxygenase (ADO) enzymes. Based on detailed kinetic and mechanistic studies, we have found further evidence for the ambiphilic behaviour of the peroxo intermediates proposed for diferric oxidoreductase enzymes.


Assuntos
Aldeído Desidrogenase/química , Materiais Biomiméticos/química , Compostos Férricos/química , Oxigênio/química , Ribonucleotídeo Redutases/química , Aldeídos/química , Cinética , Oxirredução , Fenóis/química
3.
Nat Commun ; 10(1): 2653, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201319

RESUMO

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Assuntos
Sítio Alostérico/genética , Proteínas de Bactérias/genética , Ribonucleotídeo Redutases/genética , Regulação Alostérica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/ultraestrutura , Ribonucleotídeos/metabolismo , Espalhamento a Baixo Ângulo
4.
Biochemistry ; 58(14): 1845-1860, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30855138

RESUMO

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.


Assuntos
Domínio Catalítico , Desoxirribonucleotídeos/química , Conformação Proteica , Ribonucleotídeo Redutases/química , Actinobacillus/enzimologia , Actinobacillus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Modelos Moleculares , Filogenia , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X
5.
J Biol Inorg Chem ; 24(2): 211-221, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30689052

RESUMO

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tirosina/metabolismo , Valina/metabolismo , Reagentes para Ligações Cruzadas/química , Geobacillus/enzimologia , Ferro/química , Manganês/química , Mutação Puntual , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Tirosina/química , Valina/química
6.
Biochemistry ; 58(6): 697-705, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30571104

RESUMO

Proteins forming dimers or larger complexes can be strongly influenced by their effector-binding status. We investigated how the effector-binding event is coupled with interface formation via computer simulations, and we quantified the correlation of two types of contact interactions: between the effector and its binding pocket and between protein monomers. This was achieved by connecting the protein dynamics at the monomeric level with the oligomer interface information. We applied this method to ribonucleotide reductase (RNR), an essential enzyme for de novo DNA synthesis. RNR contains two important allosteric sites, the s-site (specificity site) and the a-site (activity site), which bind different effectors. We studied these different binding states with atomistic simulation and used their coarse-grained contact information to analyze the protein dynamics. The results reveal that the effector-protein dynamics at the s-site and dimer interface formation are positively coupled. We further quantify the resonance level between these two events, which can be applied to other similar systems. At the a-site, different effector-binding states (ATP vs dATP) drastically alter the protein dynamics and affect the activity of the enzyme. On the basis of these results, we propose a new mechanism of how the a-site regulates enzyme activation.


Assuntos
Ribonucleotídeo Redutases/metabolismo , Nucleotídeos de Timina/metabolismo , Regulação Alostérica/fisiologia , Sítio Alostérico , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Multimerização Proteica/fisiologia , Ribonucleotídeo Redutases/química , Nucleotídeos de Timina/química
8.
Oncogene ; 38(13): 2364-2379, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30518875

RESUMO

DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Since inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs) that are essential for DNA replication and DNA damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to this drug during treatment. Thus, new drugs that inhibit RNR are needed to be developed. In this study, we identified a synthetic analog of resveratrol (3,5,4'-trihydroxy-trans-stilbene), termed DHS (trans-4,4'-dihydroxystilbene), that acts as a potent inhibitor of DNA replication. Molecular docking analysis identified the RRM2 (ribonucleotide reductase regulatory subunit M2) of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F-mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently decreased synthesis of dNTPs with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anti-cancer agent that targets RRM2 with therapeutic potential either alone or in combination with other agents to arrest cancer development.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Neoplasias/patologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Estilbenos/farmacologia , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Simulação de Acoplamento Molecular , Subunidades Proteicas/efeitos dos fármacos , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Estilbenos/química , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Eur J Med Genet ; 62(11): 103574, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30439532

RESUMO

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.


Assuntos
Acidose/genética , Proteínas de Ciclo Celular/genética , Morte Perinatal , Ribonucleotídeo Redutases/genética , Acidose/diagnóstico por imagem , Acidose/patologia , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação/genética , Gravidez , Conformação Proteica , Ribonucleotídeo Redutases/química
10.
EMBO Rep ; 20(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30498077

RESUMO

The murine cytomegalovirus protein M45 protects infected mouse cells from necroptotic death and, when heterologously expressed, can protect human cells from necroptosis induced by tumour necrosis factor receptor (TNFR) activation. Here, we show that the N-terminal 90 residues of the M45 protein, which contain a RIP homotypic interaction motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of the human kinases RIPK1 and RIPK3, and the Z-DNA binding protein 1 (ZBP1), to form heteromeric amyloid fibrils in vitro Mutation of the tetrad residues in the M45 RHIM attenuates homo- and hetero-amyloid assembly by M45, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. The M45 RHIM preferentially interacts with RIPK3 and ZBP1 over RIPK1 and alters the properties of the host RHIM protein assemblies. Our results indicate that M45 mimics the interactions made by RIPK1 or ZBP1 with RIPK3, thereby forming heteromeric amyloid structures, which may explain its ability to inhibit necroptosis.


Assuntos
Amiloide/metabolismo , Necroptose , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Ribonucleotídeo Redutases/metabolismo , Proteínas Virais/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Amiloidose/etiologia , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ribonucleotídeo Redutases/química , Relação Estrutura-Atividade , Proteínas Virais/química
11.
Nature ; 563(7731): 416-420, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30429545

RESUMO

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.


Assuntos
Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/metabolismo , Metais , Mycoplasma/metabolismo , Ribonucleotídeos/metabolismo , Sequência de Aminoácidos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Sistema Imunitário/metabolismo , Ferro/metabolismo , Metais/metabolismo , Modelos Moleculares , Mycoplasma/efeitos dos fármacos , Mycoplasma/enzimologia , Mycoplasma/genética , Óperon/genética , Oxirredução , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeos/química , Tirosina/química , Tirosina/metabolismo
12.
J Am Chem Soc ; 140(46): 15744-15752, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30347141

RESUMO

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the ß2 subunit (Y122•) necessary for nucleotide reductase activity. Upon binding the cognate α2 subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active α2ß2 complex from the Y122• site in ß2 to the substrate activating cysteine residue (C439) in α2 via a pathway of redox active amino acids (Y122[ß] ↔ W48[ß]? ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]) spanning >35 Å. Ionizable residues at the α2ß2 interface are essential in mediating RT, and therefore control activity. One of these mutations, E350X (where X = A, D, Q) in ß2, obviates all RT, though the mechanism of control by which E350 mediates RT remains unclear. Herein, we utilize an E350Q-photoß2 construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y122• → Y356) is replaced by direct photochemical radical generation of Y356•. These data present compelling evidence that E350 conveys allosteric information between the α2 and ß2 subunits facilitating conformational gating of RT that specifically targets Y122• reduction, while the fidelity of the remainder of the RT pathway is retained.


Assuntos
Ribonucleotídeo Redutases/química , Transporte de Elétrons , Escherichia coli/enzimologia , Radicais Livres/química , Radicais Livres/metabolismo , Modelos Moleculares , Processos Fotoquímicos , Conformação Proteica , Ribonucleotídeo Redutases/metabolismo
13.
Proc Natl Acad Sci U S A ; 115(40): 10022-10027, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224458

RESUMO

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.


Assuntos
Di-Hidroxifenilalanina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Radicais Livres/química , Ribonucleotídeo Redutases/química , Tirosina/química , Di-Hidroxifenilalanina/metabolismo , Proteínas de Escherichia coli/metabolismo , Radicais Livres/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tirosina/metabolismo
14.
Angew Chem Int Ed Engl ; 57(39): 12754-12758, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30075052

RESUMO

Proton transfer reactions are of central importance to a wide variety of biochemical processes, though determining proton location and monitoring proton transfers in biological systems is often extremely challenging. Herein, we use two-color valence-to-core X-ray emission spectroscopy (VtC XES) to identify protonation events across three oxidation states of the O2 -activating, radical-initiating manganese-iron heterodinuclear cofactor in a class I-c ribonucleotide reductase. This is the first application of VtC XES to an enzyme intermediate and the first simultaneous measurement of two-color VtC spectra. In contrast to more conventional methods of assessing protonation state, VtC XES is a more direct probe applicable to a wide range of metalloenzyme systems. These data, coupled to insight provided by DFT calculations, allow the inorganic cores of the MnIV FeIV and MnIV FeIII states of the enzyme to be assigned as MnIV (µ-O)2 FeIV and MnIV (µ-O)(µ-OH)FeIII , respectively.


Assuntos
Proteínas de Bactérias/metabolismo , Ribonucleotídeo Redutases/metabolismo , Espectrometria por Raios X , Proteínas de Bactérias/química , Chlamydia trachomatis/enzimologia , Teoria da Densidade Funcional , Compostos Férricos/química , Íons/química , Ferro/química , Manganês/química , Prótons , Ribonucleotídeo Redutases/química
15.
Structure ; 26(9): 1237-1250.e6, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30057026

RESUMO

The tumor suppressor p53 acts as a transcription factor recognizing diverse DNA response elements (REs). Previous structural studies of p53-DNA complexes revealed non-canonical Hoogsteen geometry of A/T base pairs at conserved CATG motifs leading to changes in DNA shape and its interface with p53. To study the effects of DNA shape on binding characteristics, we designed REs with modified base pairs "locked" into either Hoogsteen or Watson-Crick form. Here we present crystal structures of these complexes and their thermodynamic and kinetic parameters, demonstrating that complexes with Hoogsteen base pairs are stabilized relative to those with all-Watson-Crick base pairs. CATG motifs are abundant in p53REs such as GADD45 and p53R2 related to cell-cycle arrest and DNA repair. The high-resolution structures of these complexes validate their propensity to adopt the unique Hoogsteen-induced structure, thus providing insights into the functional role of DNA shape and broadening the mechanisms that contribute to DNA recognition by proteins.


Assuntos
DNA/química , DNA/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Elementos de Resposta , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo
16.
J Biol Chem ; 293(26): 10413-10414, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29959279

RESUMO

Ribonucleotide reductases (RNRs) are essential enzymes producing de novo deoxynucleotide (dNTP) building blocks for DNA replication and repair and regulating dNTP pools important for fidelity of these processes. A new study reveals that the class Ia Escherichia coli RNR is regulated by dATP via stabilization of an inactive α4ß4 quaternary structure, slowing formation of the active α2ß2 structure. The results support the importance of the regulatory α4ß4 complex providing insight in design of experiments to understand RNR regulation in vivo.


Assuntos
Nucleotídeos de Desoxiadenina/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Escherichia coli/enzimologia , Modelos Moleculares , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo
17.
Proc Natl Acad Sci U S A ; 115(20): E4594-E4603, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712847

RESUMO

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5'-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner.


Assuntos
Bacillus subtilis/enzimologia , Nucleotídeos de Desoxiadenina/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos de Desoxiadenina/química , Ligantes , Ligação Proteica , Conformação Proteica , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Especificidade por Substrato
18.
Biochemistry ; 57(18): 2679-2693, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29609464

RESUMO

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.


Assuntos
Flavoproteínas/química , Manganês/química , Ribonucleotídeo Redutases/química , Superóxidos/química , Catálise , Domínio Catalítico , Flavobacterium/química , Flavobacterium/enzimologia , Flavoproteínas/metabolismo , Ferro/química , Oxirredução , Oxigênio/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismo , Tirosina/química
19.
Biochemistry ; 57(24): 3402-3415, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29630358

RESUMO

3-Aminotyrosine (NH2Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH2Y of key importance for its application in mechanistic studies. By combining the tRNA/NH2Y-RS suppression technology with a model protein tailored for amino acid redox studies (α3X, X = NH2Y), the formal reduction potential of NH2Y32(O•/OH) ( E°' = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ E°' between NH2Y32(O•/OH) and Y32(O•/OH) when measured under reversible conditions is ∼300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH2Y and Y. We have also generated D6-NH2Y731-α2 of ribonucleotide reductase (RNR), which when incubated with ß2/CDP/ATP generates the D6-NH2Y731•-α2/ß2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz 1H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH2• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH2Y-RNR incorporated by infidelity of the tRNA/NH2Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.


Assuntos
Escherichia coli/enzimologia , Ribonucleotídeo Redutases/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Estrutura Molecular , Oxirredução , Ribonucleotídeo Redutases/química , Tirosina/química
20.
J Biol Chem ; 293(26): 10404-10412, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29700111

RESUMO

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a ß2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2ß2 state allowing for radical transfer from ß2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4ß4 ring-like state. Here, we investigate whether the α4ß4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-ß interface (S37D/S39A-α2, S39R-α2, S39F-α2, E42K-α2, or L43Q-α2) that disrupt the α4ß4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4ß4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-ß interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-ß interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α2 or E42K-α2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.


Assuntos
Substituição de Aminoácidos , Escherichia coli/enzimologia , Multimerização Proteica/genética , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Nucleotídeos de Desoxiadenina/farmacologia , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química
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