Class Ib Ribonucleotide Reductase Research Articles

Stability of the Tyrosyl Radical in the Ribonucleotide Reductase Beta Subunit of Arcobacter bivalviorum

Arcobacter spp., such as Arcobacter bivalviorum (A. bivalviorum), are free-living organisms found in diverse environments and associated with animals. They are considered emerging enteropathogens and potential zoonotic agents. Ribonucleotide reductase (RNR) is the key enzyme that is used to convert ribonucleotides into deoxyribonucleoside triphosphates (dNTPs). This process utilises radical-based chemistry and is crucial for DNA biosynthesis and repair. There are three RNR classes, with class I RNR the most studied, present in A. bivalviorum, eukaryotes, and many prokaryotes. Class I RNRs are further divided into three subclasses: Ia, Ib, and Ic. Class Ib RNRs use a dimanganese-oxo centre, unlike class Ia RNRs, which use a diiron-oxo centre. A. bivalviorum possesses a class Ia enzyme that requires a diferric tyrosyl radical cofactor located within its beta (β) subunit. Indeed, both the efficiency and fidelity of DNA synthesis are influenced by the stability of the tyrosyl radical (Y•) in the RNR, which is a critical aspect of its enzymatic function. This study investigates the stability of the Y-radical (Y•) site within the RNR β subunit of A. bivalviorum and the nature of the neighbouring amino acid residues. To achieve these goals, we developed a model of the RNR β subunit of A. bivalviorum, using the RNR β subunit of Aquifex aeolicus as a reference template (7aik.1. A PDB). The results provide some important details about the radical site and its surrounding residues, highlighting the influence of the protein structure on the stability of the radical. These findings may guide the development of novel inhibitors targeting this enzyme in A. bivalviorum.

Preparation and characterization of MnIIMnIII complexes with relevance to class Ib ribonucleotide reductases

The Mn2 complex [MnII2(TPDP)(O2CPh)2](BPh4) (1, TPDP = 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol, Ph =phenyl) was prepared and subsequently characterized via single-crystal X-ray diffraction, X-ray absorption, electronic absorption, and infrared spectroscopies, and mass spectrometry. 1 was prepared in order to explore its properties as a structural and functional mimic of class Ib ribonucleotide reductases (RNRs). 1 reacted with superoxide anion (O2•–) to generate a peroxido-MnIIMnIII complex, 2. The electronic absorption and electron paramagnetic resonance (EPR) spectra of 2 were similar to previously published peroxido-MnIIMnIII species. Furthermore, X-ray near edge absorption structure (XANES) studies indicated the conversion of a MnII2 core in 1 to a MnIIMnIII state in 2. Treatment of 2 with para-toluenesulfonic acid (p-TsOH) resulted in the conversion to a new MnIIMnIII species, 3, rather than causing O—O bond scission, as previously encountered. 3 was characterized using electronic absorption, EPR, and X-ray absorption spectroscopies. Unlike other reported peroxido-MnIIMnIII species, 3 was capable of oxidative O—H activation, mirroring the generation of tyrosyl radical in class Ib RNRs, however without accessing the MnIIIMnIV state.

Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases in Mycobacteria

Class I ribonucleotide reductases consisting of α and β subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the β subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the β subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen Mycobacterium tuberculosis and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidised and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the β subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the M. tuberculosis NrdF2I complex. A density close to Tyr 110 at a distance <2.3 Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the β subunit of the ribonucleotide reductase complex in M. tuberculosis.

A New Thiolate-Bound Dimanganese Cluster as a Structural and Functional Model for Class Ib Ribonucleotide Reductases.

In class Ib ribonucleotide reductases (RNRs) a dimanganese(II) cluster activates superoxide (O2 ⋅- ) rather than dioxygen (O2 ), to access a high valent MnIII -O2 -MnIV species, responsible for the oxidation of tyrosine to tyrosyl radical. In a biomimetic approach, we report the synthesis of a thiolate-bound dimanganese complex [MnII 2 (BPMT)(OAc)2 ](ClO)4 (BPMT=(2,6-bis{[bis(2-pyridylmethyl)amino]methyl}-4-methylthiophenolate) (1) and its reaction with O2 ⋅- to form a [(BPMT)MnO2 Mn]2+ complex 2. Resonance Raman investigation revealed the presence of an O-O bond in 2, while EPR analysis displayed a 16-line St =1/2 signal at g=2 typically associated with a MnIII MnIV core, as detected in class Ib RNRs. Unlike all other previously reported Mn-O2 -Mn complexes, generated by O2 ⋅- activation at Mn2 centers, 2 proved to be a capable electrophilic oxidant in aldehyde deformylation and phenol oxidation reactions, rendering it one of the best structural and functional models for class Ib RNRs.

Ein neuer Thiolat‐gebundener Dimangan‐Cluster als strukturelles und funktionales Modell der Class Ib Ribonukleotidreduktasen

AbstractIn Class Ib Ribonukleotidreduktasen (RNRs) aktiviert ein Dimangan(II)‐Cluster Superoxid (O2⋅−) anstelle von molekularem Sauerstoff (O2), um eine hochvalente MnIII‐O2‐MnIV‐Spezies zu bilden, die für die Oxidation von Tyrosin zum Tyrosyl‐Radikal verantwortlich ist. Im vorliegenden biomimetischen Ansatz wird ein Thiolat‐gebundener Dimangan‐Komplex [MnII2(BPMT)(OAc)2](ClO)4 (BPMT=(2,6‐Bis{[bis(2‐pyridylmethyl)amino]methyl}‐4‐methylthiophenolat) (1) synthetisiert und dessen Reaktion mit O2⋅− zur Bildung des [(BPMT)MnO2Mn]2+‐Komplexes 2 gezeigt. Resonanz‐Raman‐Untersuchungen zeigen das Vorliegen einer O‐O‐Bindung in 2, während die ESR‐Analyse ein Signal bei g=2 mit 16 Linien für St= aufzeigt, das typischerweise mit einem MnIIIMnIV‐Kern assoziiert wird, wie es auch in Class Ib RNR detektiert wurde. Anders als zuvor publizierte Mn‐O2‐Mn‐Komplexe, die durch O2⋅−‐Aktivierung an Mn2‐Zentren gebildet wurden, ist 2 ein geeignetes elektrophiles Oxidationsmittel für Aldehyd‐Deformylierungsreaktionen und Phenol‐Oxidationsreaktionen, sodass es eines der besten strukturellen und funktionalen Modelle der Class Ib RNRs darstellt.

Rv0233 is not essential for the survival of Mycobacterium tuberculosis in stress conditions.

Mycobacterium tuberculosis is an important global pathogen. We were interested in understanding the role of Rv0233, a proposed subunit of the class IB ribonucleotide reductase, and its role in surviving stress conditions. We constructed an in-frame, unmarked deletion strain of M. tuberculosis and characterized its growth and survival under replicating or non-replicating conditions. We confirmed previous studies that found that Rv0233 is not essential for aerobic growth or survival in the presence of nitrite. We demonstrated that the deletion of Rv0233 does not affect susceptibility to frontline tuberculosis drugs or hydrogen peroxide. The deletion strain survived equally well under nutrient starvation or in hypoxia and was not attenuated for growth in macrophages.

Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII.

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallocofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction. The proteins required for MnIII2-Y• cluster formation in class Ib RNRs are NrdF (β-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both Streptococcus sanguinis and Escherichia coli MnII2-NrdF structures have a constriction in the channel immediately above the metal site. In E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine-to-threonine substitution was investigated using S. sanguinis and Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of MnIII2-Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1µM and the previously reported preference for MnII could not be explained by affinity only.

Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties.

Low‐molecular‐weight (low M r) thioredoxin reductases (TrxRs) are homodimeric NADPH‐dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx‐like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR‐like ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant‐type FNR. In Bacillus cereus, a TrxR‐like FNR has been shown to reduce the flavodoxin‐like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR and lack the homologous TrxR‐like FNR. Due to the structural similarity between TrxRs and TrxR‐like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low M r TrxR may be able to replace TrxR‐like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity toward NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co‐substrate binding in TrxR.

The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron

Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving–Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with MnII and FeII in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving–Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.Graphical

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

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.

Transcriptomic analysis of Bifidobacterium longum subsp. longum BBMN68 in response to oxidative shock

Bifidobacterium longum strain BBMN68 is sensitive to low concentrations of oxygen. A transcriptomic study was performed to identify candidate genes for B. longum BBMN68’s response to oxygen treatment (3%, v/v). Expression of genes and pathways of B. longum BBMN68 involved in nucleotide metabolism, amino acid transport, protein turnover and chaperones increased, and that of carbohydrate metabolism, translation and biogenesis decreased to adapt to the oxidative stress. Notably, expression of two classes of ribonucleotide reductase (RNR), which are important for deoxyribonucleotide biosynthesis, was rapidly and persistently induced. First, the class Ib RNR NrdHIEF was immediately upregulated after 5 min oxygen exposure, followed by the class III RNR NrdDG, which was upregulated after 20 min of exposure. The upregulated expression of branched-chain amino acids and tetrahydrofolate biosynthesis-related genes occurred in bifidobacteria in response to oxidative stress. These change toward to compensate for DNA and protein damaged by reactive oxygen species (ROS). In addition, oxidative stress resulted in improved B. longum BBMN68 cell hydrophobicity and autoaggregation. These results provide a rich resource for our understanding of the response mechanisms to oxidative stress in bifidobacteria.

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.

Ribonucleotide Reductases from Bifidobacteria Contain Multiple Conserved Indels Distinguishing Them from All Other Organisms: In Silico Analysis of the Possible Role of a 43 aa Bifidobacteria-Specific Insert in the Class III RNR Homolog.

Bifidobacteria comprises an important group/order of bacteria whose members have widespread usage in the food and health industry due to their health-promoting activity in the human gastrointestinal tract. However, little is known about the underlying molecular properties that are responsible for the probiotic effects of these bacteria. The enzyme ribonucleotide reductase (RNR) plays a key role in all organisms by reducing nucleoside di- or tri- phosphates into corresponding deoxyribose derivatives required for DNA synthesis, and RNR homologs belonging to classes I and III are present in either most or all Bifidobacteriales. Comparative analyses of these RNR homologs have identified several novel sequence features in the forms of conserved signature indels (CSIs) that are exclusively found in bifidobacterial RNRs. Specifically, in the large subunit of the aerobic class Ib RNR, three CSIs have been identified that are uniquely found in the Bifidobacteriales homologs. Similarly, the large subunit of the anaerobic class III RNR contains five CSIs that are also distinctive characteristics of bifidobacteria. Phylogenetic analyses indicate that these CSIs were introduced in a common ancestor of the Bifidobacteriales and retained by all descendants, likely due to their conferring advantageous functional roles. The identified CSIs in the bifidobacterial RNR homologs provide useful tools for further exploration of the novel functional aspects of these important enzymes that are exclusive to these bacteria. We also report here the results of homology modeling studies, which indicate that most of the bifidobacteria-specific CSIs are located within the surface loops of the RNRs, and of these, a large 43 amino acid insert in the class III RNR homolog forms an extension of the allosteric regulatory site known to be essential for protein function. Preliminary docking studies suggest that this large CSI may be playing a role in enhancing the stability of the RNR dimer complex. The possible significances of the identified CSIs, as well as the distribution of RNR homologs in the Bifidobacteriales, are discussed.

Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST).

This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus ( Lofstad et al., 2016 ). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.

Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.

To reduce ribonucleotides to deoxyribonucleotides, the manganese-bound form of class Ib ribonucleotide reductase (RNR) must be activated via a pathway that involves redox protein(s). The reduced flavoprotein NrdI is an important protein in this pathway, as it reduces dioxygen to superoxide. Superoxide then reacts with the RNR Mn(II)2 site to generate a tyrosyl radical that is required for catalysis. A native NrdI reductase has not yet been identified. We herein demonstrate through kinetic and spectroscopic studies that an endogenous flavodoxin reductase can function as the NrdI reductase in Bacillus cereus. When the flavodoxin reductase reduces NrdI, tyrosyl radical formation in RNR is promoted under aerobic conditions, significantly increasing the radical yield. Thus, a missing piece of the class Ib RNR NrdI redox pathway has finally been identified.

Structure of B. cereus Class Ib Ribonucleotide Reductase NrdI-Fe2-NrdF Complex

Class Ib ribonucleotide reductases (RNRs) use a dimetal-tyrosyl radical (Y·) cofactor in their NrdF (beta2) subunit to initiate ribonucleotide reduction in the NrdE (beta2) subunit. Contrary to the diferric tyrosyl radical (FeIII2-Y·) cofactor, which can self-assemble from FeII2-NrdF and O2, generation of the MnIII2-Y· cofactor requires the reduced form of a flavoprotein, NrdIhq, and O2 for its assembly. Here we report the 1.8 Å resolution crystal structure of Bacillus cereus Fe2-NrdF in complex with NrdI. Compared to the previously solved Escherichia coli NrdI-MnII2-NrdF structure, NrdI and NrdF binds similarly in Bacillus cereus through conserved core interactions. This protein-protein association seems to be unaffected by metal ion type bound in the NrdF subunit. The Bacillus cereus MnII2-NrdF and Fe2-NrdF structures, also presented here, show conformational flexibility of residues surrounding the NrdF metal ion site. The movement of one of the metal-coordinating carboxylates is linked to the metal type present at the di-metal site, and not associated with NrdI-NrdF binding. This carboxylate conformation seems to be vital for the water network connecting the NrdF di-metal site and the flavin in NrdI. From these observations, we suggest that metal-dependent variations in carboxylate coordination geometries are important for active Y· cofactor generation in class Ib RNRs. Additionally, we show that binding of NrdI to NrdF would structurally interfere with the suggested alfa2beta2 (NrdE-NrdF) holoenzyme formation, suggesting the potential requirement for NrdI dissociation before NrdE-NrdF assembly after NrdI-activation. The mode of interactions between the proteins involved in the class Ib RNR system is, however, not fully resolved. [1]

The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y·) or a dimanganese-Y· cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe 2 (III) -Y· cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe 2 (III) -Y· cofactor. We also report enzymatic activity of Fe 2 (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

Streptococcus sanguinis Class Ib Ribonucleotide Reductase: HIGH ACTIVITY WITH BOTH IRON AND MANGANESE COFACTORS AND STRUCTURAL INSIGHTS

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/β2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.