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Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the ß2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-ß interface that restricts the ability of ß2 to obtain a position that is capable of radical transfer. ATP breaks the α-ß interface, freeing ß2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for ß. When ATP displaces dATP, the helix rewinds, dismantling the α-ß interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.
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Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O and C-N bond breaking and formation steps that are otherwise challenging for nonradical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here, we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.
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Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Microbiota , Acetiltransferases/química , Acetiltransferases/metabolismo , Anaerobiose , Bactérias/química , Proteínas de Bactérias/química , Carboxiliases/química , Carboxiliases/metabolismo , Fermentação , Humanos , Ligases/química , Ligases/metabolismo , Modelos Moleculares , Conformação Proteica , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismoRESUMO
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.
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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/químicaRESUMO
The anaerobic gut microbial pathway that converts choline into trimethylamine (TMA) is broadly linked to human disease. Here, we describe the discovery that betaine aldehyde inhibits TMA production from choline by human gut bacterial isolates and a complex gut community. In vitro assays and a crystal structure suggest betaine aldehyde targets the gut microbial enzyme choline TMA-lyase (CutC). In our system, we do not observe activity for the previously reported CutC inhibitor 3,3-dimethylbutanol (DMB). The workflow we establish for identifying and characterizing betaine aldehyde provides a framework for developing additional inhibitors of gut microbial choline metabolism, including therapeutic candidates.
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Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Colina/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Anaerobiose/efeitos dos fármacos , Domínio Catalítico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) take advantage of the ribosomal translation machinery to generate linear peptides that are subsequently modified with heterocycles and/or macrocycles to impose three-dimensional structure and thwart degradation by proteases. Although RiPP precursors are limited to proteinogenic amino acids, post-translational modifications (PTMs) can alter the structure of individual amino acids and thereby improve the stability and biological activity of the molecule. These "tailoring modifications" often occur on amino acid side chains-for example, hydroxylation, methylation, halogenation, prenylation, and acylation-but can also take place within the backbone, as in epimerization, or can result in capping of the N- or C-terminus. At one extreme, these modifications can be essential to the activity of the RiPP, either as a compulsory step in reaching the final molecule or by imparting chemical functionality required for biological activity. At the other extreme, tailoring PTMs may have little effect on the activity in an in vitro setting-possibly because of test conditions that do not match the biological context in which the PTMs evolved. Establishing the molecular basis for the function of tailoring PTMs often requires a three-dimensional structure of the RiPP bound to its biological target. These structures have revealed roles for tailoring PTMs that include providing additional hydrogen bonds to targets, rigidifying the RiPP structure to reduce the entropic cost of binding, or altering the secondary structure of the peptide backbone. Bacterial RiPPs are particularly suited to structural characterization, as they are relatively easy to isolate from laboratory cultures or to produce in a heterologous host. The identification of new tailoring PTMs within bacteria is also facilitated by clustering of the genes encoding tailoring enzymes with those of the RiPP precursor and primary modification enzymes. In this Account, we describe the effects of tailoring PTMs on RiPP structure, their interactions with biological targets, and their influence on RiPP stability, with a focus on bacterial RiPP classes. We also discuss the enzymes that generate tailoring PTMs and highlight examples of and prospects for engineering of RiPPs.
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Produtos Biológicos/metabolismo , Ribossomos/metabolismo , Acilação , Halogênios/química , Hidroxilação , Metilação , Peptídeos/química , Peptídeos/metabolismo , Prenilação , Conformação Proteica , Estabilidade ProteicaRESUMO
The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism.
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Neisseria/enzimologia , Substâncias Redutoras/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tiorredoxinas/metabolismo , Aminoácidos/metabolismo , Biocatálise , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Citidina Trifosfato/metabolismo , Citosina/metabolismo , Dissulfetos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Biológicos , NADP , Oxirredução , Ribonucleotídeo Redutases/química , Thermotoga maritima/enzimologia , Fatores de TempoRESUMO
Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits ß and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded ß/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits ß and γ display folds related to high potential iron-sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSß binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSß, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSß in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations.
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Proteínas de Bactérias/química , Carbono-Carbono Liases/química , Proteínas Ferro-Enxofre/química , Thauera/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Radicais Livres/química , Glicina/química , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C-C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSß, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these "X-succinate synthases" and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes.
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Proteínas de Bactérias/química , Carbono-Carbono Liases/química , Thauera/enzimologia , Tolueno/química , Domínio Catalítico , Estrutura Quaternária de ProteínaRESUMO
Fluorinated tyrosines (FnY's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y·) in the ß subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in ß and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-ß and the X-ray structures of each resulting ß with a diferric cluster are reported and compared with wt-ß2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-ß2, Fe(2+), and O2 to produce â¼1 Y·/ß2 and â¼3 Fe(3+)/ß2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-ß2. Each FnY·-ß2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the (19)F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·'s. The variability of the abiotic FnY pKa's (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·'s in catalysis and to investigate the importance and identity of hopping Y·'s within redox active proteins proposed to protect them from uncoupled radical chemistry.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Exorribonucleases/química , Flúor/química , Methanocaldococcus/enzimologia , Ribonucleotídeo Redutases/química , Tirosina/química , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Catálise , Simulação por Computador , Cristalização , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Radicais Livres/química , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Methanocaldococcus/genética , Modelos Moleculares , Oxirredução , Oxigênio/química , Fosforilação , RNA de Transferência/química , TemperaturaRESUMO
Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α(2)) and the radical-generation subunit (ß(2)) interact. Here we present the first structure of a complex between α(2) and ß(2) subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α(4)ß(4) ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α(2)ß(2) configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α(2)ß(2) and α(4)ß(4) species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α(2)ß(2) and α(4)ß(4) entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.
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Escherichia coli/enzimologia , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Ribonucleotídeo Redutases/química , Regulação Alostérica/fisiologia , Cristalização , Cristalografia por Raios X , DNA/biossíntese , Microscopia Eletrônica , UltracentrifugaçãoRESUMO
Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.
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Highlights from the Science family of journals.