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 Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

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 E0(RSSR/RSSR˙-) reduction potential has been determined for a homologous series of disulfides. The electrochemical potentials are found to be strongly dependent on the structures and electronic properties of the substituents of the disulfides. In the case of cysteine, a standard potential of E0(RSSR/RSSR˙-) = -1.38 V vs. NHE is determined, making the disulfide radical anion of cysteine one of the most reducing cofactors in biology.

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 Escherichia coli class Ia RNR requires two homodimeric subunits, α and ß. The active form is an asymmetric αα'ßß' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C439•), and the ß subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[ß] ↔ W48?[ß] ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[ß] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/ß interface. An E52[ß] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[ß]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[ß], R331[α], E326[α], and E326[α'] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) 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 Ng 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). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4ß4 state in the presence of the ß subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

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 Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by 17O 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 17O high-frequency electron-nuclear double resonance (ENDOR) in conjunction with H217O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α2ß2 E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···17O distances in the range 2.8-3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.

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

The class Ia ribonucleotide reductase of Escherichia coli requires strict regulation of long-range radical transfer between two subunits, α and ß, through a series of redox-active amino acids (Y122•[ß] ↔ W48?[ß] ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y356 is regulated by proton release involving a specific residue, E52[ß], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent. An E52Q variant is incapable of Y356 oxidation via the native radical transfer pathway or non-native photochemical oxidation, following photosensitization by covalent attachment of a photo-oxidant at position 355[ß]. Substitution of Y356 for various FnY analogues in an E52Q-photoß2, where the side chain remains deprotonated, recovered photochemical enzymatic turnover. Transient absorption and emission data support the conclusion that Y356 oxidation requires E52 for proton management, suggesting its essential role in gating radical transport across the protein-protein interface.

Conformational Motions and Water Networks at the α/ß Interface in E. coli 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 E. coli Ia RNR is composed of two homodimers that form the active α2ß2 complex. Catalysis is initiated by long-range radical translocation over a ∼32 Å proton-coupled electron transfer (PCET) pathway involving Y356ß and Y731α at the interface. Resolving the PCET pathway at the α/ß interface has been a long-standing challenge due to the lack of structural data. Herein, molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α2ß2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.

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. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

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 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.

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 Escherichia coli class Ia RNR is an asymmetric α2ß2 complex in which α2 contains the active site and ß2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ß2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of ß2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:ß2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ß reveals that the rate constants for subunit association (163 ± 7 µM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ß2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:ß2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4ß4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

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 Escherichia coli for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of the C439U α2 mutant protein with nucleotide substrates and the cognate ß2 subunit demonstrates that the endogenous Y122• of ß2 is reduced under turnover conditions, presumably through radical transfer to form a transient U439• species. This putative U439• species is formed in a kinetically competent fashion but is incapable of initiating nucleotide reduction via 3'-H abstraction. An analogous C225U α2 protein is also capable of radical transfer from Y122•, but the radical-based substrate chemistry partitions between turnover and stalled reduction akin to the reactivity of mechanism-based inhibitors of RNR. The results collectively demonstrate the essential role of cysteine redox chemistry in the class I RNRs and establish a new tool for investigating thiyl radical reactivity in biology.

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. 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.

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 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.

Basis of dATP inhibition of RNRs.

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.

An endogenous dAMP ligand in Bacillus subtilis 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. 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.