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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 β subunit to render a chimeric RNR. Inspired by a recent cryo- EM structure, a [Re] photooxidant is located adjacent to Y356[β], 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 β metallocofactor highlights the adaptability of the 10- stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multistep proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ∼35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask examination of the kinetics of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET), and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively "tight" subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits. [ABSTRACT FROM AUTHOR]

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaCCc) is a highly soluble protein with a turnover number of 75 s-1 and no lag phase in coenzyme A (CoA) release. Studies with [1-14C]HBCoA and PhaCCc monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [3H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaCCc with [1-14C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS-PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s-1. We propose that the [3H]sDCoA forms via attack of CoA on the oxoester of the [3H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaCCc, and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation. [ABSTRACT FROM AUTHOR]

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 FeIIIFeIII-tyrosyl radical (Y••) cofactor (class Ia), a MnIIIMnIII-Y•• cofactor (class Ib), and a MnIVFeIII 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. [ABSTRACT FROM AUTHOR]

Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)2-Y•] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O2. Apo-rNrdF loaded using 4 Mn(II)/β2, O2, and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y• [Mn(III)2-Y•] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)2-Y• and Fe(III)2-Y• rNrdF generate dCDP at rates of 132 and 10 nmol min-1 mg-1, respectively (both normalized for 1 Y•/β2). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)2-Y• center with 2 Mn/β2 and 0.5 Y•/β2 and an activity of 318-363 nmol min-1 mg-1 (normalized for 1 Y•/β2). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)2-Y• enzyme. [ABSTRACT FROM AUTHOR]

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active anhim (n, m = 2 or 6) complexes. a binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (Ki = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (Ki = 17 nM; t1/2 = 23 min). CDP protects a from its inhibition. The altered off-rate of ClFDP from E • ClFDP' by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its (site binding. Size exclusion chromatography of hRNR or a alone with ClFDP or ClFTP, ±ATP or dGTP, reveals in each case that a forms a kinetically stable hexameric state. This is the first example of hexamerization of a induced by an NDP analog that reversibly binds at the active site. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Ycan be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Ynganese(III)-Ys raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms’ growth and virulence. [ABSTRACT FROM AUTHOR]

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (FeIII2-Y∙) and dimanganese(III)-Y∙ (MnIII2-Y∙) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y∙ cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with 95% purity, with 0.2 Y∙/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min-1 mg-1. Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII2-Y∙ cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosyleobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynuclèotides and is rapidly (<30 s) inactivated by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). [l-3H]. and [5-3H]F2CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RN Rand that 0.71 equiv of eytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixlure of [I-3H]F2CTPand [l-2HIF2CTP followed by reduction with NaBH4, alkylation with iodoaeetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass speetrometry (MS) of a 3H/2H-labeled peptide containing C731 and C736 from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two eysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [l'-3H]F2CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH4 subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These sludieS and the ones in the preceding paper (DOI: l0.1021/bi9021318) allow proposal of a mechanism of inactivation of RN R by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs. [ABSTRACT FROM AUTHOR]

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is expressed under iron-limited and oxidative Stress conditions. This RNR is composed of two homodimeric subunits: α2 (NrdE), where nucleotide reduction occurs, and β2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)6-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with MnII and incubated with fully reduced NrdI (NrdIhq) and O2. Active RNR was rapidly produced with 0.25 ± 0.03 tyrosyl radical (Y·) per β2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is MnIII2-Y·, which we propose is generated by MnII2-NrdF reacting with two equivalents of HO2-, produced by reduction of O2 by NrdF-bound NrdIhq. In the absence of NrdIhq, with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with FeII and incubated with O2 in the presence or absence of NrdIhq gave 0.2 and 0.7 Y·/β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdIhq hinders FeIII2-Y· cofactor assembly in vitro. We propose that Nrdl is an essential player in E. coli class Ib RNR cluster assembly and that the MnIII2-Y· cofactor, not the diferric-Y· one, is the active metallocofactor in vivo. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: α2 and β2. The α subunit contains the site of nucleotide reduction, and the β subunit contains the essential diferric-tyrosyl radical (Y•) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (α) and NrdB (β), is expressed under normal aerobic growth conditions. The class lb RNR, composed of NrdE (α) and NrdF (β), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y• levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y• maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class lb RNR. It possesses redox properties unprecedented for a flavodoxin (Eox/sq = 264 ± 17 mV and Esq/hq = -255 ± 17 my) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE. [ABSTRACT FROM AUTHOR]

This review focuses on nontemplate-dependent polymerases that use water-soluble substrates and convert them into water-insoluble polymers that form granules or inclusions within the cell. The initial part of the review summarizes briefly the current knowledge of polymer formation catalyzed by starch and glycogen synthases, polyphosphate kinase (a polymerase), cyanophycin synthetases, and rubber synthases. Specifically, our current understanding of their mechanisms of initiation, elongation (including granule formation), termination, remodeling, and polymer re-utilization will be presented. General underlying principles that govern these types of polymerization reactions will be enumerated as a paradigm for all nontemplate-dependent polymerizations. The bulk of the review then focuses on polyhydroxyalka-noate (PHA) synthases that generate polyoxoesters. These enzymes are of interest as they generate biodegradable polymers. Our current knowledge of PHA production and utilization in vitro and in vivo as well as the contribution of many proteins to these processes will be reviewed. [ABSTRACT FROM AUTHOR]

Abstract: Time-resolved absorption spectroscopy has been employed to study the directionality and rate of charge transfer in W–Y and Ac-W–Y dipeptides as a function of pH. Excitation with 266-nm nanosecond laser pulses produces both W⋅ (or [⋅WH]+, depending on pH) and Y⋅. Between pH 6 and 10, W⋅ to was found to oxidize Y with kX⋅=9.0×104 s−1 and 1.8×104 s−1 for the W–Y and Ac-W–Y dipeptide systems, respectively. The intramolecular charge transfer rate increases as the pH is lowered over the range 6>pH>2. For 10

Bleomycins are a family of glycopeptide antibiotics that have potent antitumour activity against a range of lymphomas, head and neck cancers and germ-cell tumours. The therapeutic efficacy of the bleomycins is limited by development of lung fibrosis. The cytotoxic and mutagenic effects of the bleomycins are thought to be related to their ability to mediate both single-stranded and double-stranded DNA damage, which requires the presence of specific cofactors (a transition metal, oxygen and a one-electron reductant). Progress in understanding the mechanisms involved in the therapeutic efficacy of the bleomycins and the unwanted toxicity and elucidation of the biosynthetic pathway of the bleomycins sets the stage for developing a more potent, less toxic therapeutic agent. [ABSTRACT FROM AUTHOR]

DNA strand breaks can result as a direct or indirect consequence of oxidative damage to the nucleic acid bases and/or deoxyribose sugars. Ionizing radiation and the antitumor agents, the bleomycins (BLMs) and enediynes, share in common the ability to indirectly cause DNA strand scission after C4' hydrogen atom abstraction from the deoxyribose moiety. In the case of extensively studied BLMs, the C4' radical generated under anaerobic conditions results in production of a 4'-keto abasic site after C4' oxidation to a cation and H2O addition. To study the structure, stability, and repair of this lesion, a general method is reported for its homogeneous preparation in any sequence context. 4'-Azido-2'-deoxyuridine- 5'-triphosphate is incorporated into duplex DNA using a primer, a template containing a restriction enzyme (NgoM IV) cleavage site at its 3'-end, and HTV-l reverse transcriptase. The two strands of the duplex are separated based on size after cleavage with the restriction enzyme. The single-stranded (ss) DNA containing 4'-azido-2'-deoxyuridine, when treated with uracil-DNA glycosylase, results in quantitative release of uracil, azide, and generation of a ss-DNA containing the 4'-keto abasic site. This lesion is characterized directly by MALDI-TOF MS and indirectly by subsequent reduction, enzymatic digestion, and GCIMS. The stability of duplex DNA containing a 4'-keto abasic site relative to an abasic site iii the same sequence context is reported under physiological conditions. [ABSTRACT FROM AUTHOR]

The bleomycins (BLMs) are used clinically in combination chemotherapy, their clinical usefulness being limited by the accompanying pulmonary toxicity. Much has been learned about the structure and function of BLMs in vitro. However, the mechanism of their cytoxicity in vivo, including their target(s), metal cofactor(s) effecting nucleic acid cleavage and its (their) oxidation state, concentrations of BLM in the nucleus of the cell, BLM metabolism, hot spots for double-strand DNA cleavage, and their repair, have remained elusive. New methods offer new opportunities to revisit and solve old problems, which could ultimately lead to development of a more effective therapeutic. [Copyright &y& Elsevier]

Discusses the mechanism of long-range electron transfer (ET) and proton-coupled electron transfer (PCET) in biology, using the radical initiation process of class I ribonucleotide reductases (RNR) as a focal point. Theoretical framework of ET and PCET; Principles of ET; Methods to study radical initiation in RNR.

New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction. [Copyright &y& Elsevier]

Examines the mechanisms of couples phasin synthesis with the presence of polyhydroxyalkanoates (PHA). Effects of Ralstonia eutropha Pha on the regulation PHA phasin accumulation; Emergence of phasins during PHA synthesis; Description of polyhydroxyalkanoates.

Presents insight to the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. Generation of purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain; Outcome of the studies with the phaP deletion strain; Affectivity of PhaP to PHB synthesis under growth conditions.

Focuses on a study on the evolution of ribonucleotide reduction. Background and mechanisms of ribonucleotide reductases. Details on active-site structures; Relationship between allosteric specificity site and activity-site structures.

Deals with class-I ribonucleotide reductases (RNR), for which the Escherichia coli enzyme serves as a prototype. Formation of the diiron(III)-tyrosyl-radical cofactor of class-I RNRs; Other diiron(III) requiring proteins; Function of the tyrosyl radical; Importance of tyrosine radicals or modified tyrosine radicals in metabolism.

Studies the principles that govern the formation of protein radicals and their roles in catalysis. Involvement of non-amino acid in enzymatic systems; Chemical properties of enzymes; Conversion of ribonucleotide reductases; Detection of the thiyl radical.

Presents a study on the description of the successful efforts in providing evidence that the nitrogen centered radical is covalently bound to the sulfur of a cysteine of the R1 subunit of E. coli ribonucleotide reductase (RNR) using electron paramagnetic resonance (EPR) spectroscopy together with RNR containing [beta-2H]cysteine.

Presents a study which reveal that F2dCTP is shown to be a soichiometric mechanism-based inactivator of the adenosylcobalamin- (AdoCbl-) dependent Lactobacillus leichmannii ribonucleoside triphosphate reductase (RTPR). Analysis of the fate of the F2dCTP and AdoCbl; Materials and methods used in the study; Results of the study.

Investigates the interaction of the dioxygenase thymine hydroxylase with 5-ethynyluracil. Covalent modification of the enzyme; Tandem mass spectrometry of the enzyme adduct.

Abstract: Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is a 1:1 complex of two homodimeric subunits: α2 and β2. As a first step towards mapping the subunit interface, β2 (V365C) was labeled with [14C]-benzophenone (BP) iodoacetamide. The resulting [14C]-BP–β2 (V365C) was complexed with α2 and irradiated at 365nm for 30min at 4°C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [14C]-labeled peptides were identified: two contained a peptide in β to which the BP was attached. The third contained the same β peptide and a peptide in α found in its αD helix. These results provide direct support for the proposed docking model of α2β2. [Copyright &y& Elsevier]

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, a and b. Because of the transient subunit association, an atomic resolution structure of an active a2b2 RNR complex has been elusive. We used a doubly substituted b2, E52Q/(2,3,5)-trifluorotyrosine122-b2, to trap wild-type a2 in a long-lived a2b2 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. [ABSTRACT FROM AUTHOR]

The article features scientist Chris Raetz, who was born in East Berlin, Germany in 1946 and died on August 16, 2011 because of cancer. It discusses Raetz's profession in chemistry at Harvard Medical School in Boston, Massachusetts with Eugene Kennedy, in which he studied the enzymes of lipid metabolism. It also highlights his expertise in other fields including biochemistry, structural biology, and genetics.

In this article the author discusses aspects of a study within the issue regarding biochemistry research. Topics include a brief overview of radicals, which are highly energetic molecular species, such as atoms, the role of radical-based reactions in metabolism, and the influence of the cellular compound S-adenosylmethionine (SAM) in a novel radical chemical reaction the enables ribosome methylation in bacteria.

The author reflects on the role of enzyme. She discusses that genomes have revealed thousands of DNA base sequences and the roles of genes that have been incorrectly assigned in genome databases. She added a study that established the function of protein Tm0936 found in Thermotoga maritima bacteria which belongs to the amidohydrolase superfamily (AHS) of enzymes. Furthermore, the author emphasizes the reaction that Tm0936 catalyses that could form undiscovered degradation pathway.

The article focuses on the study about the forward and reverse electron transfer with the Y356dihydroxyphenylalanine (DOPA)-beta2 Heterodimer of Escherichia coli ribonucleotide reductases (RNRs). Results show that the amounts of DOPA suggest that the interaction of the alpha2/beta2 complex is asymmetric. In addition, results also constitutes the first direct observation of reverse electron transfer between residues 356 and 122 in the beta subunit of RNR.

The article focuses on the specific site replacement of Y356 with 3,4- Dihydroxyphenylalanine in the β2 subunit of Escherichia coli ribonucleotide reductase. Ribonucleotide reductases (RNR) catalyze the conversion of nucleotides to deoxynucleotides in all organism. α2 houses the site for nucleotide reduction that control substrate specificity rates. β2 contains the diferric-tyrosyl radical cofactor, which is essential for the radical-dependent reduction process.

Pays tribute to biochemist Robert H. Abeles, who died on June 18, 2000. Contribution to mechanistic enzymology; Personal and professional background of Abeles; Experiments designed by Abeles.

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 (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:β2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122β ↔ [W48β] ↔ Y356β ↔ Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:β2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns -- μs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 X 10 s~ when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport. [ABSTRACT FROM AUTHOR]

Reports the first purification of the enzymes required to synthesize microcin B17, an enzyme-based macromolecular machine inside the bacterium Escherichia coli. Report by Li et al. on page 1188 of the November 15, 1996 issue of `Science'; The ability of such machines to synthesize a diverse array of medicinally interesting polypeptides and polyketides with unusual ring structures; Information on the ring structures; Possible implications of the study.

Discusses how two cofactors, adenosylcobalamin and methylcobalamin, participate in enzyme catalyzed transformations and the biosynthetic pathway of vitamin B12. Crystallization of Escherichia coli methionine synthase; Role of S-adenosylmethionine in methylation; Structure of B12-requiring enzyme.

Commentary. Presents information as it relates to the use of ribonucleotide reductases (RNRs). Details on the application of RNRs; Reference to the use of RNRs as catalysts; Findings of studies conducted.

Activation of the diferrous center of the ß2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied 57Fe, 14,15N, 17O, and ¹H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of 17O and ¹H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH-)FeIII-O-FeIV]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the 14N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238. [ABSTRACT FROM AUTHOR]

Investigates the steps involved in DNA biosynthesis. Role of the ribonucleotide reductase (RNR) enzyme from Escherichia coli; Location of the cysteines in the active site of the R1 subunit; Cysteines proposed to play a part in nucleotide reduction.

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 α4β4 state in the presence of the β 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. [ABSTRACT FROM AUTHOR]

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (MnIII2-Y.) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted MnIII2-Y. NrdF using dithiothreitol as the reductant, however, was low (160 nmol min-1 mg-1). The apparent tight affinity between the two subunits, distinct from all class la RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystailographic characterization of an "active" RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from MnIII2-Y. NrdF, yielding enzyme containing 4 Mn and 1 Y./β2. Goal two was achieved by cloning expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min-1 mg-1 using a 1:1 mixture of NrdE:MnIII2-Y. NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1.) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 μM), NrdE is a monomer (a) and MnIII2-Y. NrdF is a dimer (β2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations. [ABSTRACT FROM AUTHOR]

The reversible Y-O•/Y-OH redox properties of the α,Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at oH values below the apparent pK (8.0 ±0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°'(Y- O•/Y-OH) of 1026 ± 4 mV versus the normal hydrogen electrode (pH 5.70 ± 0.02) and shows that the fluoro substitutions lower the E°' by -30 ± 3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues. [ABSTRACT FROM AUTHOR]

Chris Raetz passed away on August 16,2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gramnegative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpin-nings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiositydriven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues. [ABSTRACT FROM AUTHOR]

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y·) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric-tyrosyl radical (Y122O•) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O• reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O• was studied using reaction-induced FT-IR spectroscopy and [13C]aspartate-labeled β2. These Y122O• minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2. [ABSTRACT FROM AUTHOR]

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 W48[?] ⇆ Y356 in β2, across the subunit interface to Y731 ⇆ Y730 ⇆ C439 in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2, and this mutant was incubated with β2, cytidine 5′-diphosphate, and adenosine 5′-triphosphate to generate a NH2Y730• in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H⋯O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available. [ABSTRACT FROM AUTHOR]

Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β2 subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(FeIII 2-Y.)(FeIII 2)] cofactor for de novoDNAbiosynthesis. Severalmechanismsfor3-APinhibition of β2 have been proposed; one involves direct iron chelation fromβ2, whereas a second involvesY. destruction by reactive oxygen species formed in situ in the presence ofO2 andreductant by Fe(II)-(3-AP). Inactivation of β2 can thus arise from cofactor destruction by loss of iron or Y.. In vitro kinetic data on the rates of 55Fe and Y. loss from [(55FeIII 2-Y.)(55FeIII 2)]-β2 under aerobic and anaerobic conditions reveal that Y. loss alone is sufficient for rapidβ2 inactivation. OxyblotTM and mass spectrometric analyses of trypsin-digested inhibited β2, and lack of Y. loss from H2O2 and O2-. treatment together preclude reactive oxygen species involvement in Y. loss. Three mammalian cell lines treated with 5 μM 3-AP reveal Y. loss and β2 inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(FeIII 2-Y.)(FeIII 2)]-β2 in lysates, similar iron-content in β2 immunoprecipitated from 3-AP-treated and untreated [55Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β2 are most consistent with Y. loss being the predominant mode of inhibition, with β2 remaining iron-loaded and stable. A modelconsistent with in vitroandcell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y. of the [(FeIII 2-Y.)(FeIII 2)] cofactor ofβ2 [ABSTRACT FROM AUTHOR]