RÉSUMÉ
The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy.
Sujet(s)
Protéines de Caenorhabditis elegans/composition chimique , Caenorhabditis elegans/enzymologie , Mutation , Thymidine kinase/composition chimique , Thymidine kinase/génétique , Animaux , Antiviraux/composition chimique , Antiviraux/métabolisme , Caenorhabditis elegans/métabolisme , Protéines de Caenorhabditis elegans/génétique , Domaine catalytique , Humains , Cinétique , Spécificité du substrat , Zidovudine/composition chimique , Zidovudine/métabolismeRÉSUMÉ
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)â¢) 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)â¢â W(48)â Y(356) in ß2 to Y(731)â Y(730)â 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)Yâ¢) 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.
Sujet(s)
Substitution d'acide aminé , Escherichia coli/enzymologie , Nucléotides/biosynthèse , Ribonucleotide reductases/génétique , Ribonucleotide reductases/métabolisme , Tyrosine/analogues et dérivés , Biocatalyse , Radicaux libres/métabolisme , Cinétique , Modèles moléculaires , Nucléotides/composition chimique , Conformation des protéines , Ribonucleotide reductases/composition chimique , Ribonucleotide reductases/isolement et purification , Analyse spectrale , Tyrosine/composition chimiqueRÉSUMÉ
Escherichia coli ribonucleotide reductase is an α2ß2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(â¢)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(â¢)âW(48)âY(356) within ß to Y(731)âY(730)âC(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in ß2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(â¢)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-ß2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(â¢) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(â¢)/ß2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(â¢) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(â¢) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(â¢) is predominantly located at 356 in ß2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-ß2, a mechanism for PCET uncoupling in NO(2)Y(â¢)-RNR is proposed.
Sujet(s)
Oxydants/composition chimique , Ribonucleotide reductases/composition chimique , Tyrosine/analogues et dérivés , Absorption , Adénosine triphosphate/métabolisme , Biocatalyse , Diphosphates/métabolisme , Spectroscopie de résonance de spin électronique , Transport d'électrons , Escherichia coli/enzymologie , Radicaux libres/composition chimique , Cinétique , Modèles moléculaires , Conformation des protéines , Protons , Ribonucleotide reductases/métabolisme , Tyrosine/composition chimiqueRÉSUMÉ
E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5'-diphosphates into 2'-deoxynucleotides and is composed of two subunits: alpha2 and beta2. During turnover, a stable tyrosyl radical (Y*) at Y(122)-beta2 reversibly oxidizes C(439) in the active site of alpha2. This radical propagation step is proposed to occur over 35 A, to use specific redox-active tyrosines (Y(122) and Y(356) in beta2, Y(731) and Y(730) in alpha2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO(2)Y, pK(a) 7.1) has been incorporated in place of Y(122), Y(731), and Y(730) to probe how the protein environment perturbs each pK(a) in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 x 10(-3) that of the wild-type (wt) subunit. The [NO(2)Y(730)]-alpha2 and [NO(2)Y(731)]-alpha2 each exhibit a pK(a) of 7.8-8.0 with E and E/beta2. The pK(a) of [NO(2)Y(730)]-alpha2 is elevated to 8.2-8.3 in the S/E/beta2 complex, whereas no further perturbation is observed for [NO(2)Y(731)]-alpha2. Mutations in pathway residues adjacent to the NO(2)Y that disrupt H-bonding minimally perturb its pK(a). The pK(a) of NO(2)Y(122)-beta2 alone or with alpha2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO(2)Y]-alpha2 mutants (2.1-3.1 A resolution), which show minimal structural perturbation compared to wt-alpha2. Together with the pK(a) of the previously reported NO(2)Y(356)-beta2 (7.5 in the alpha2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541-14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway.
Sujet(s)
Domaine catalytique , Ribonucleotide reductases/composition chimique , Ribonucleotide reductases/métabolisme , Tyrosine/analogues et dérivés , Biocatalyse , Escherichia coli/enzymologie , Concentration en ions d'hydrogène , Modèles moléculaires , Oxydoréduction , Liaison aux protéines , Sous-unités de protéines/composition chimique , Sous-unités de protéines/métabolisme , ARN de transfert/métabolisme , Spécificité du substrat , Tyrosine/métabolismeRÉSUMÉ
The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli. No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.
Sujet(s)
Ribonucleotide reductases/composition chimique , Salmonella typhimurium/enzymologie , Tyrosine/composition chimique , Sites de fixation , Cristallographie aux rayons X , Spectroscopie de résonance de spin électronique , Escherichia coli/enzymologie , Radicaux libres , Fer/composition chimique , Ligands , Modèles statistiques , Conformation des protéines , SpectrophotométrieRÉSUMÉ
Ribonucleotide reductase is an indispensable enzyme for all cells, since it catalyses the biosynthesis of the precursors necessary for both building and repairing DNA. The ribonucleotide reductase class I enzymes, present in all mammals as well as in many prokaryotes and DNA viruses, are composed mostly of two homodimeric proteins, R1 and R2. The reaction involves long-range radical transfer between the two proteins. Here, we present the first crystal structure of a ribonucleotide reductase R1/R2 holocomplex. The biological relevance of this complex is based on the binding of the R2 C terminus in the hydrophobic cleft of R1, an interaction proven to be crucial for enzyme activity, and by the fact that all conserved amino acid residues in R2 are facing the R1 active sites. We suggest that the asymmetric R1/R2 complex observed in the 4A crystal structure of Salmonella typhimurium ribonucleotide reductase represents an intermediate stage in the reaction cycle, and at the moment of reaction the homodimers transiently form a tight symmetric complex.
Sujet(s)
Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Structure quaternaire des protéines , Ribonucleotide reductases/composition chimique , Ribonucleotide reductases/métabolisme , Salmonella typhimurium/enzymologie , Séquence d'acides aminés , Protéines bactériennes/génétique , Sites de fixation , Cristallographie aux rayons X , Dimérisation , Modèles moléculaires , Données de séquences moléculaires , Structure moléculaire , Complexes multiprotéiques , Ribonucleotide reductases/génétique , Alignement de séquencesRÉSUMÉ
Two nrdF genes of Mycobacterium tuberculosis code for different R2 subunits of the class Ib ribonucleotide reductase (RNR). The proteins are denoted R2F-1 and R2F-2 having 71% sequence identity. The R2F-2 subunit forms the biologically active RNR complex with the catalytic R1E-subunit. We present the structure of the reduced R2F-2 subunit to 2.2 A resolution. Comparison of the R2F-2 structure with a model of R2F-1 suggests that the important differences are located at the C-terminus. We found that within class Ib, the E-helix close to the iron diiron centre has two preferred conformations, which cannot be explained by the redox-state of the diiron centre. In the R2F-2 structure, we also could see a mobility of alphaE in between the two conformations.
Sujet(s)
Mycobacterium tuberculosis/enzymologie , Ribonucleotide reductases/composition chimique , Séquence d'acides aminés , Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Cristallographie aux rayons X/méthodes , Modèles moléculaires , Conformation des protéines , Structure secondaire des protéines , Sous-unités de protéines/composition chimique , Ribonucleotide reductases/métabolismeRÉSUMÉ
The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.
Sujet(s)
Ribonucleotide reductases/composition chimique , Ribonucleotide reductases/métabolisme , Salmonella typhimurium/enzymologie , Régulation allostérique/physiologie , Site allostérique , Séquence d'acides aminés , Sites de fixation , Cristallographie aux rayons X , Cystéine/composition chimique , Cystéine/métabolisme , Nucléotide désoxyadenylique/composition chimique , Nucléotide désoxyadenylique/métabolisme , Nucléotides désoxycytidyliques/composition chimique , Nucléotides désoxycytidyliques/métabolisme , Dimérisation , Escherichia coli/enzymologie , Structures macromoléculaires , Modèles moléculaires , Données de séquences moléculaires , Oxydoréduction , Conformation des protéines , Sous-unités de protéines/composition chimique , Sous-unités de protéines/métabolisme , Similitude de séquences d'acides aminés , Nucléotides thymidyliques/composition chimique , Nucléotides thymidyliques/métabolismeRÉSUMÉ
The nrdE gene product R1E, the large subunit of the class 1b Salmonella typhimurium ribonucleotide reductase, has been overexpressed, purified and crystallized. Initially, the protein crystallized in two orthorhombic space groups, C222(1) and P2(1)2(1)2, using tartrate and PEG 6000 as precipitants, respectively. Better diffracting crystals belonging to the tetrahedral space group P4(3)2(1)2 were obtained using sodium malonate as precipitant. The P4(3)2(1)2 crystals could only be obtained after seeding from a drop containing C222(1) crystals grown in sodium tartrate. Thus, streak-seeding resulted in crystals of a supergroup to C222(1). Data to 2.8 A resolution have been collected on the P4(3)2(1)2 crystals which contained one R1E subunit in the asymmetric unit.