RESUMEN

As ribonucleotide reductase (RNR) plays a crucial role in nucleic acid metabolism, it is an important target for anticancer therapy. The thiosemicarbazone Triapine is an efficient R2 inhibitor, which has entered ∼20 clinical trials. Thiosemicarbazones are supposed to exert their biological effects through effectively binding transition-metal ions. In this study, six iminodiacetate-thiosemicarbazones able to form transition-metal complexes, as well as six dicopper(II) complexes, were synthesized and fully characterized by analytical, spectroscopic techniques (IR, UV-vis; 1H and 13C NMR), electrospray ionization mass spectrometry, and X-ray diffraction. The antiproliferative effects were examined in several human cancer and one noncancerous cell lines. Several of the compounds showed high cytotoxicity and marked selectivity for cancer cells. On the basis of this, and on molecular docking calculations one lead dicopper(II) complex and one thiosemicarbazone were chosen for in vitro analysis as potential R2 inhibitors. Their interaction with R2 and effect on the Fe(III)2-Y· cofactor were characterized by microscale thermophoresis, and two spectroscopic techniques, namely, electron paramagnetic resonance and UV-vis spectroscopy. Our findings suggest that several of the synthesized proligands and copper(II) complexes are effective antiproliferative agents in several cancer cell lines, targeting RNR, which deserve further investigation as potential anticancer drugs.

Asunto(s)

Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos Organometálicos/farmacología , Ribonucleótido Reductasas/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cobre/química , Cobre/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Iminoácidos/química , Iminoácidos/farmacología , Ratones , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Relación Estructura-Actividad , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Células Tumorales Cultivadas

RESUMEN

Escherichia coli ribonucleotide reductase is an α2ß2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in ß2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in ß2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of ß. The resulting protein, Y(356)NH(2)Y-ß2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

Asunto(s)

Sustitución de Aminoácidos , Escherichia coli/enzimología , Nucleótidos/biosíntesis , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Tirosina/análogos & derivados , Biocatálisis , Radicales Libres/metabolismo , Cinética , Modelos Moleculares , Nucleótidos/química , Conformación Proteica , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/aislamiento & purificación , Análisis Espectral , Tirosina/química

RESUMEN

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 (Fe(III)(2)-Y(•)) and dimanganese(III)-Y(•) (Mn(III)(2)-Y(•)) cofactors in the ß2 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 Mn(III)(2)-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(•)/ß2, 0.9 Mn/ß2, 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 Mn(III)(2)-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.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Ribonucleótido Reductasas/química , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Manganeso , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Tirosina/química , Tirosina/metabolismo

RESUMEN

Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated gamma-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 A, alpha = 90, beta = 106.7, gamma = 90 degrees. The data set collected was 95.3% complete, with an R(merge) of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%.

Asunto(s)

Herpesvirus Humano 8/enzimología , Ribonucleótido Reductasas/química , Cristalización , Cristalografía por Rayos X , Expresión Génica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación

RESUMEN

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP 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 RNR and that 0.71 equiv of cytosine 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 mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.

Asunto(s)

Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Citidina Trifosfato/análogos & derivados , Inhibidores Enzimáticos/química , Lactobacillus leichmannii/enzimología , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Alquilación , Secuencia de Aminoácidos , Cobamidas/química , Cobamidas/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Citosina Desaminasa/antagonistas & inhibidores , Citosina Desaminasa/síntesis química , Inhibidores Enzimáticos/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Ribonucleótido Reductasas/aislamiento & purificación , Espectrometría de Masas en Tándem

RESUMEN

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5'-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, alpha and beta, for activity. Humans possess two beta subunits: one involved in S phase DNA replication (beta) and a second in mitochondrial DNA replication (beta' or p53R2) and potentially DNA repair. Gemcitabine (F(2)C) is used clinically as an anticancer agent, and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with alpha (specific activity of 400 nmol min(-1) mg(-1)) and beta' (0.6 Y./beta'2 and a specific activity of 420 nmol min(-1) mg(-1)) establishes that F(2)CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-(3)H]-F(2)CDP or [5-(3)H]-F(2)CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv of tightly associated cytosine to alpha2. SDS-PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of alpha migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1'-(3)H]-F(2)CDP with active site mutants C444S/A, C218S/A, and E431Q/D-alpha and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of alpha and that, in the case of C218S-alpha, alpha migrates as a 90 kDa protein. Analysis of the inactivated wt-alpha/beta' RNR by size exclusion chromatography indicates a quaternary structure of alpha6beta'6. A mechanism of inactivation common with halpha/beta is presented.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Citidina Difosfato/análogos & derivados , Inhibidores Enzimáticos/toxicidad , Ribonucleótido Reductasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/aislamiento & purificación , Cromatografía en Gel , Citidina Difosfato/química , Citidina Difosfato/toxicidad , Daño del ADN/genética , Reparación del ADN/genética , Inhibidores Enzimáticos/química , Humanos , Mutagénesis Sitio-Dirigida , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Ribonucleótido Reductasas/fisiología

RESUMEN

Human ribonucleotide reductase (RR) small subunits, M2 and P53R2, play key roles in forming RR holoenzyme and supplying nucleotide precursors for DNA replication and repair. Currently, we are studying the redox property, structure, and function of hRRM2 and p53R2. In the cell-free system, p53R2 did not oxidize a reactive oxygen species (ROS) indicator Carboxy-H(2)DCFDA, but hRRM2 did. Further studies demonstrated that purified recombinant p53R2 protein has the catalase activity to scavenge H(2)O(2). Over-expression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas over-expression of hRRM2 did not result in the collapse of mitochondrial membrane potential. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its enzymatic activity.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Subunidades de Proteína/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Fluoresceínas/metabolismo , Depuradores de Radicales Libres/metabolismo , Vectores Genéticos/genética , Humanos , Peróxido de Hidrógeno/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/enzimología , Oxidación-Reducción/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Ribonucleósido Difosfato Reductasa/aislamiento & purificación , Ribonucleótido Reductasas/aislamiento & purificación

RESUMEN

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: alpha2 and beta2. The alpha subunit contains the site of nucleotide reduction, and the beta 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 (alpha) and NrdB (beta), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (alpha) and NrdF (beta), 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 Ib RNR. It possesses redox properties unprecedented for a flavodoxin (E(ox/sq) = -264 +/- 17 mV and E(sq/hq) = -255 +/- 17 mV) 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.

Asunto(s)

Coenzimas/metabolismo , Escherichia coli/enzimología , Flavodoxina/metabolismo , Hierro/metabolismo , Ribonucleótido Reductasas/metabolismo , Tirosina/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Flavodoxina/química , Flavodoxina/genética , Flavodoxina/aislamiento & purificación , Radicales Libres/metabolismo , Expresión Génica , Oxidación-Reducción , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Análisis Espectral , Especificidad por Sustrato , Volumetría , Tirosina/análogos & derivados

RESUMEN

The SRL4 (YPL033C) gene was initially identified by the screening of Saccharomyces cerevisiae genes that play a role in DNA metabolism and/or genome stability using the SOS system of Escherichia coli. In this study, we found that the srl4Delta mutant cells were resistant to the chemicals that inhibit nucleotide metabolism and evidenced higher dNTP levels than were observed in the wild-type cells in the presence of hydroxyurea. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16 degrees C) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30 degrees C. Furthermore, srl4Delta was shown to suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. These results indicate that SRL4 may be involved in the regulation of dNTP production by its function as a negative regulator of ribonucleotide reductase.

Asunto(s)

Proteínas de Ciclo Celular/genética , Desoxirribonucleótidos/metabolismo , Mutación , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Frío , Daño del ADN , Regulación Fúngica de la Expresión Génica , Hidroxiurea/farmacología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Temperatura , Rayos Ultravioleta

RESUMEN

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that Trp5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain, as compared to Trp5.

Asunto(s)

Antituberculosos , Inhibidores Enzimáticos , Mycobacterium tuberculosis/enzimología , Péptidos , Ribonucleótido Reductasas/antagonistas & inhibidores , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Clonación Molecular , Técnicas Químicas Combinatorias/métodos , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Reacción en Cadena de la Polimerasa/métodos , Relación Estructura-Actividad Cuantitativa , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Estereoisomerismo

RESUMEN

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439.) in R1 over a distance of 35 A, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y.s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.

Asunto(s)

Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Ribonucleótido Reductasas/química , Tirosina/química , Dimerización , Escherichia coli/enzimología , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Radicales Libres/análisis , Cinética , Modelos Químicos , Subunidades de Proteína/química , Ribonucleótido Reductasas/clasificación , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Soluciones/química

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Subunidades de Proteína/fisiología , Ribonucleótido Reductasas/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Humanos , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación

RESUMEN

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.

Asunto(s)

Proteínas Algáceas/metabolismo , Euglena gracilis/enzimología , Proteínas Protozoarias/metabolismo , Ribonucleótido Reductasas/metabolismo , Vitamina B 12/metabolismo , Proteínas Algáceas/clasificación , Proteínas Algáceas/genética , Proteínas Algáceas/aislamiento & purificación , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleótido Reductasas/clasificación , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Alineación de Secuencia

RESUMEN

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The enzyme is composed of two subunits: R1 and R2. R1 contains the active site for nucleotide reduction and the allosteric effector sites that regulate the specificity and turnover rate. R2 contains the diferric-tyrosyl (Y(*)) radical cofactor that initiates nucleotide reduction by a putative long-range proton-coupled electron transfer (PCET) pathway over 35 A. This pathway is thought to involve specific amino acid radical intermediates (Y122 to W48 to Y356 within R2 to Y731 to Y730 to C439 within R1). In an effort to study radical initiation, R2 (375 residues) has been synthesized semisynthetically. R2 (residues 1-353), attached to an intein and a chitin binding domain, was constructed, and the protein was expressed (construct 1). This construct was then incubated with Fe(2+) and O(2) to generate the diferric-Y(*) cofactor, and the resulting protein was purified using a chitin affinity column. Incubation of construct 1 with 2-mercaptoethanesulfonic acid (MESNA) resulted in the MESNA thioester of R2 (1-353) (construct 2). A peptide containing residues 354-375 of R2 was generated using solid-phase peptide synthesis where 354, a serine in the wild-type (wt) R2, was replaced by a cysteine. Construct 2 and this peptide were ligated, and the resulting full-length R2 was separated from truncated R2 by anion-exchange chromatography. The purified protein had a specific activity of 350 nmol min(-1) mg(-1), identical to the same protein generated by site-directed mutagenesis when normalized for Y(*). As a first step in studying the radical initiation by PCET, R2 was synthesized with Y356 replaced by 3-nitrotyrosine (NO(2)Y). The protein is inactive (specific activity 1 x 10(-4) that of wt-R2), which permitted a determination of the pK(a) of the NO(2)Y in the R1/R2 complex in the presence of substrate and effectors. Under all conditions, the pK(a) was minimally perturbed. This has important mechanistic implications for the radical initiation process.

Asunto(s)

Proteínas de Escherichia coli/biosíntesis , Radicales Libres/química , Iniciación de la Cadena Peptídica Traduccional , Fragmentos de Péptidos/biosíntesis , Ribonucleótido Reductasas/biosíntesis , Tirosina/análogos & derivados , Tirosina/química , Secuencia de Aminoácidos , Quitina/metabolismo , Electroquímica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Ésteres , Hidrólisis , Cinética , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Iniciación de la Cadena Peptídica Traduccional/genética , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Plásmidos , Estructura Terciaria de Proteína/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tirosina/genética

RESUMEN

The gene for ribonucleotide reductase from Anabaena sp. strain PCC 7120 was identified and expressed in Escherichia coli. This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein. The intein splices itself from the protein when it is expressed in E. coli, yielding an active ribonucleotide reductase of 765 residues. The mature enzyme was purified to homogeneity from E. coli extracts. Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography. The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector. The enzyme is absolutely dependent on the addition of the cofactor, 5'-adenosylcobalamin. These properties are characteristic of the class II-type reductases. The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii. In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp. strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes.

Asunto(s)

Anabaena/genética , Escherichia coli/enzimología , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Vitamina B 12/metabolismo , Secuencia de Aminoácidos , Anabaena/enzimología , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

Activation of plasminogen (plg) to plasmin by the staphylococcal activator, staphylokinase (SAK), is effectively regulated by the circulating inhibitor, alpha2-antiplasmin (alpha2AP). Here it is demonstrated that intact Staphylococcus aureus cells and solubilized staphylococcal cell wall proteins not only protected SAK-promoted plg activation against the inhibitory effect of alpha2AP but also enhanced the activation. The findings suggest that the surface-associated plg activation by SAK may have an important physiological function in helping staphylococci in tissue dissemination. Amino acid sequencing of tryptic peptides originating from the 59-, 56- and 43-kDa proteins, isolated as putative plg-binding proteins, identified them as staphylococcal inosine 5'-monophosphate dehydrogenase, alpha-enolase, and ribonucleotide reductase subunit 2, respectively.

Asunto(s)

Proteínas Bacterianas/metabolismo , Metaloendopeptidasas/metabolismo , Plasminógeno/metabolismo , Staphylococcus aureus/metabolismo , alfa 2-Antiplasmina/farmacología , Secuencia de Aminoácidos , Antifibrinolíticos/farmacología , Pared Celular/química , Pared Celular/metabolismo , Activación Enzimática/fisiología , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/metabolismo , IMP Deshidrogenasa/aislamiento & purificación , IMP Deshidrogenasa/metabolismo , IMP Deshidrogenasa/farmacología , Datos de Secuencia Molecular , Fosfopiruvato Hidratasa/metabolismo , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Ribonucleótido Reductasas/farmacología

Asunto(s)

Glutatión/análogos & derivados , Glutatión/metabolismo , Ribonucleótidos/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo , Tiorredoxinas/metabolismo , Animales , Técnicas In Vitro , Oxidación-Reducción , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Trypanosomatina/metabolismo

Asunto(s)

Pyrococcus furiosus/enzimología , Ribonucleótido Reductasas/aislamiento & purificación , Regulación Alostérica , Secuencia de Aminoácidos , Catálisis , Cromatografía Liquida/métodos , Estabilidad de Enzimas , Datos de Secuencia Molecular , Ribonucleótido Reductasas/clasificación , Ribonucleótido Reductasas/metabolismo , Homología de Secuencia de Aminoácido , Temperatura

RESUMEN

Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.

Asunto(s)

Lactococcus lactis/enzimología , Ribonucleótido Reductasas/química , Proteínas Virales/química , Adenosina Trifosfato/metabolismo , Catálisis , Cromatografía en Agarosa , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Iones , Magnesio/farmacología , Magnetismo , Modelos Químicos , Potasio/farmacología , Unión Proteica , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Factores de Tiempo , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismo

RESUMEN

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

Asunto(s)

Microtúbulos/metabolismo , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/fisiología , Huso Acromático/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Extractos Celulares/química , Centrosoma/metabolismo , Interfase , Masculino , Mitosis , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/metabolismo , Óvulo/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleótido Reductasas/aislamiento & purificación , Homología de Secuencia , Espermatozoides/metabolismo , Tubulina (Proteína)/metabolismo , Xenopus , Proteínas de Xenopus