Conserved electron donor complex Dre2-Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis.
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ABSTRACT:
SUBMITTER: Zhang Y
PROVIDER: S-EPMC4035922 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.
| S-EPMC4703083 | biostudies-literature
Project description:Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1-R2 and R1-p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4-6 times higher than that of the Trx1 system. For both complexes, the V max values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1-p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.
| S-EPMC6709626 | biostudies-literature
Project description:Ribonucleotide reductase (RNR) regulates DNA synthesis and repair in all organisms. The mechanism of Escherichia coli RNR requires radical transfer over a proton-coupled electron transfer (PCET) pathway spanning ∼32 Å across two protein subunits. A key step along this pathway is the interfacial PCET reaction between Y356 in the β subunit and Y731 in the α subunit. Herein, this PCET reaction between two tyrosines across an aqueous interface is explored with classical molecular dynamics and quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The simulations suggest that the water-mediated mechanism involving double proton transfer through an intervening water molecule is thermodynamically and kinetically unfavorable. The direct PCET mechanism between Y356 and Y731 becomes feasible when Y731 is flipped toward the interface and is predicted to be approximately isoergic with a relatively low free energy barrier. This direct mechanism is facilitated by the hydrogen bonding of water to both Y356 and Y731. These simulations provide fundamental insights into radical transfer across aqueous interfaces.
| S-EPMC10344599 | biostudies-literature
Project description:An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.
| S-EPMC6854502 | biostudies-literature
Project description:Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122(•)) that generates a thiyl radical (C439(•)) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y(•)-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s(-1)). Rapid freeze-quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y(•)-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356(•) and 0.5 equiv of dCDP, both at 30 s(-1). In the absence of an external reducing system, Y356(•) reduction occurs concomitant with F3Y reoxidation (0.4 s(-1)) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s(-1)) is observed prior to steady-state turnover (1.7 s(-1)). The [Y356(•)] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122(•) is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.
| S-EPMC4678968 | biostudies-literature
Project description:Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R1(2)R2(2) heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.
| S-EPMC2900304 | biostudies-literature
Project description:Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of alpha(2) and beta(2) subunits. Rnr1, the alpha subunit, contains regulatory and catalytic sites; Rnr2, the beta subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from Saccharomyces cerevisiae: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1-Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.
| S-EPMC1389703 | biostudies-literature
Project description:The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.
| S-EPMC3466523 | biostudies-literature
Project description:Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and is critical for DNA synthesis and repair in all organisms. Its mechanism requires radical transfer along a ∼32 Å pathway through a series of proton-coupled electron transfer (PCET) steps. Previous simulations suggested that a glutamate residue (E623) mediates the PCET reaction between two stacked tyrosine residues (Y730 and Y731) through a proton relay mechanism. This work focuses on the adjacent PCET reaction between Y730 and a cysteine residue (C439). Quantum mechanical/molecular mechanical free energy simulations illustrate that when Y730 and Y731 are stacked, E623 stabilizes the radical on C439 through hydrogen bonding with the Y730 hydroxyl group. When Y731 is flipped away from Y730, a water molecule stabilizes the radical on C439 through hydrogen bonding with Y730 and lowers the free energy barrier for radical transfer from Y730 to C439 through electrostatic interactions with the transferring hydrogen but does not directly accept the proton. These simulations indicate that the conformational motions and electrostatic interactions of the tyrosines, cysteine, glutamate, and water strongly impact the thermodynamics and kinetics of these two coupled PCET reactions. Such insights are important for protein engineering efforts aimed at altering radical transfer in RNR.
| S-EPMC9197590 | biostudies-literature
Project description:The β2 subunit of class Ia ribonucleotide reductases (RNR) contains an antiferromagnetically coupled μ-oxo bridged diiron cluster and a tyrosyl radical (Y122•). In this study, an ultraviolet resonance Raman (UVRR) difference technique describes the structural changes induced by the assembly of the iron cluster and by the reduction of the tyrosyl radical. Spectral contributions from aromatic amino acids are observed through UV resonance enhancement at 229 nm. Vibrational bands are assigned by comparison to histidine, phenylalanine, tyrosine, tryptophan, and 3-methylindole model compound data and by isotopic labeling of histidine in the β2 subunit. Reduction of the tyrosyl radical reveals Y122• Raman bands at 1499 and 1556 cm(-1) and Y122 Raman bands at 1170, 1199, and 1608 cm(-1). There is little perturbation of other aromatic amino acids when Y122• is reduced. Assembly of the iron cluster is shown to be accompanied by deprotonation of histidine. A p(2)H titration study supports the assignment of an elevated pK for the histidine. In addition, structural perturbations of tyrosine and tryptophan are detected. For tryptophan, comparison to model compound data suggests an increase in hydrogen bonding and a change in conformation when the iron cluster is removed. pH and (2)H(2)O studies imply that the perturbed tryptophan is in a low dielectric environment that is close to the metal center and protected from solvent exchange. Tyrosine contributions are attributed to a conformational or hydrogen-bonding change. In summary, our work shows that electrostatic and conformational perturbations of aromatic amino acids are associated with metal cluster assembly in RNR. These conformational changes may contribute to the allosteric effects, which regulate metal binding.
| S-EPMC3164833 | biostudies-literature