2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor NCDP.

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species.

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor NCDP.

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme.

Disulfide radical anion as a super-reductant in biology and photoredox chemistry.

Disulfides are involved in a broad range of radical-based synthetic organic and biochemical transformations. In particular, the reduction of a disulfide to the corresponding radical anion, followed by S-S bond cleavage to yield a thiyl radical and a thiolate anion plays critical roles in radical-based photoredox transformations and the disulfide radical anion in conjunction with a proton donor, mediates the enzymatic synthesis of deoxynucleotides from nucleotides within the active site of the enzyme, ribonucleotide reductase (RNR). To gain fundamental thermodynamic insight into these reactions, we have performed experimental measurements to furnish the transfer coefficient from which the standard (RSSR/RSSR˙) reduction potential has been determined for a homologous series of disulfides.

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex.

F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. class Ia RNR is an αβ enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β, nucleotide reduction in α. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant ) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR).

Radicals in Biology: Your Life Is in Their Hands.

Radicals in biology, once thought to all be bad actors, are now known to play a central role in many enzymatic reactions. Of the known radical-based enzymes, ribonucleotide reductases (RNRs) are pre-eminent as they are essential in the biology of all organisms by providing the building blocks and controlling the fidelity of DNA replication and repair. Intense examination of RNRs has led to the development of new tools and a guiding framework for the study of radicals in biology, pointing the way to future frontiers in radical enzymology.

Statistical analysis of ENDOR spectra.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing.

Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by O Electron-Nuclear Double Resonance Spectroscopy.

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate O high-frequency electron-nuclear double resonance (ENDOR) in conjunction with HO-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the αβ ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···O distances in the range 2.

Gated Proton Release during Radical Transfer at the Subunit Interface of Ribonucleotide Reductase.

The class Ia ribonucleotide reductase of requires strict regulation of long-range radical transfer between two subunits, α and β, through a series of redox-active amino acids (Y•[β] ↔ W?[β] ↔ Y[β] ↔ Y[α] ↔ Y[α] ↔ C[α]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y is regulated by proton release involving a specific residue, E[β], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent.

Conformational Motions and Water Networks at the α/β Interface in Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of Ia RNR is composed of two homodimers that form the active αβ complex. Catalysis is initiated by long-range radical translocation over a ∼32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface.

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs.

Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex.

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 β. Because of the transient subunit association, an atomic resolution structure of an active α2β2 RNR complex has been elusive.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of class Ia RNR is an asymmetric αβ complex in which α contains the active site and β contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation.

Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of the CU α mutant protein with nucleotide substrates and the cognate β subunit demonstrates that the endogenous Y of β is reduced under turnover conditions, presumably through radical transfer to form a transient U species.

Convergent allostery in ribonucleotide reductase.

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.

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β subunit (Y) necessary for nucleotide reductase activity. Upon binding the cognate α 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 αβ complex from the Y site in β to the substrate activating cysteine residue (C) in α via a pathway of redox active amino acids (Y[β] ↔ W[β]? ↔ Y[β] ↔ Y[α] ↔ Y[α] ↔ C[α]) spanning >35 Å. Ionizable residues at the αβ interface are essential in mediating RT, and therefore control activity.

Basis of dATP inhibition of RNRs.

Ribonucleotide reductases (RNRs) are essential enzymes producing 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 RNR is regulated by dATP via stabilization of an inactive α4β4 quaternary structure, slowing formation of the active α2β2 structure. The results support the importance of the regulatory α4β4 complex providing insight in design of experiments to understand RNR regulation .

An endogenous dAMP ligand in class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP.

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.

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm.

3-Aminotyrosine (NHY) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NHY of key importance for its application in mechanistic studies. By combining the tRNA/NHY-RS suppression technology with a model protein tailored for amino acid redox studies (αX, X = NHY), the formal reduction potential of NHY(O/OH) ( E°' = 395 ± 7 mV at pH 7.

3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α:β subunit interface.