Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase (original) (raw)
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Ribonucleotide Reductases: Divergent Evolution of an Ancient Enzyme
Journal of Molecular Evolution, 2002
Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their threedimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.
Acta Crystallographica Section A Foundations of Crystallography, 2002
and Functional Genomics respects: first, they all employ free radical chemistry Stockholm University through protein-based radicals, but the nature and Stockholm S-106 91 mechanism of generation of these radicals varies greatly Sweden among the three classes [2]; second, it currently appears that, given one set of physiological conditions, only one molecular species of RNR is responsible for the reduction of all four ribonucleotides. This necessitates a pre-Summary cise system of control of substrate specificity in response to the concentrations of deoxyribonucleoside Background: The specificity of ribonucleotide reductriphosphates (dNTPs) in the cell because balanced tases (RNRs) toward their four substrates is governed dNTP pools are a prerequisite for efficient and accurate by the binding of deoxyribonucleoside triphosphates DNA synthesis and repair. Imbalances in dNTP pools (dNTPs) to the allosteric specificity site. Similar patterns have been implicated in diseases through increased in the kinetics of allosteric regulation have been a strong rates of mutation [3]. argument for a common evolutionary origin of the three RNRs are governed by a sensitive allosteric mechaotherwise widely divergent RNR classes. Recent strucnism whereby the dNTP products regulate the rate of tural information settled the case for divergent evolution; reduction of the nucleoside di-or triphosphates. Two however, the structural basis for transmission of the separate allosteric functions have been localized to two allosteric signal is currently poorly understood. A comstructurally distinct sites, commonly called the specificparative study of the conformational effects of the bindity and activity sites, first by biochemical assays [4, 5] ing of different effectors has not yet been possible; in and then by X-ray crystallography for class Ia, aerobic addition, only one RNR class has been studied. enzymes [6]. Overall activity is upregulated by ATP and downregulated by dATP through the binding of these Results: Our presentation of the structures of a class effectors to the activity site [7], except in class Ib [8] III anaerobic RNR in complex with four dNTPs allows a and all but one currently known class II RNRs [9], which full comparison of the protein conformations. Discrimilack overall activity regulation. In most cases the excepnation among the effectors is achieved by two side tions are correlated to the lack of a 50 residue N-terminal chains, Gln-114 and Glu-181, from separate monomers. sequence containing amino acids critical for ATP/dATP Large conformational changes in the active site (loop binding [6]. The regulation of substrate specificity in 2), in particular Phe-194, are induced by effector binding.
Biochemical Society Transactions, 2012
RNRs (ribonucleotide reductases) are key players in nucleic acid metabolism, converting ribonucleotides into deoxyribonucleotides. As such, they maintain the intracellular balance of deoxyribonucleotides to ensure the fidelity of DNA replication and repair. The best-studied RNR is the class Ia enzyme from Escherichia coli, which employs two subunits to catalyse its radical-based reaction: β2 houses the diferric-tyrosyl radical cofactor, and α2 contains the active site. Recent applications of biophysical methods to the study of this RNR have revealed the importance of oligomeric state to overall enzyme activity and suggest that unprecedented subunit configurations are in play. Although it has been five decades since the isolation of nucleotide reductase activity in extracts of E. coli, this prototypical RNR continues to surprise us after all these years.
A Glycyl Radical Site in the Crystal Structure of a Class III Ribonucleotide Reductase
Science, 1999
Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.
Journal of Molecular Biology, 2006
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 4 Å 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.
Structure, function, and mechanism of ribonucleotide reductases
Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 2004
Ribonucleotide reductase (RNR) is the enzyme responsible for the conversion of ribonucleotides to 2V-deoxyribonucleotides and thereby provides the precursors needed for both synthesis and repair of DNA. In the recent years, many new crystal structures have been obtained of the protein subunits of all three classes of RNR. This review will focus upon recent structural and spectroscopic studies, which have offered deeper insight to the mechanistic properties as well as evolutionary relationship and diversity among the different classes of RNR. Although the three different classes of RNR enzymes depend on different metal cofactors for the catalytic activity, all three classes have a conserved cysteine residue at the active site located on the tip of a protein loop in the centre of an a/h-barrel structural motif. This cysteine residue is believed to be converted into a thiyl radical that initiates the substrate turnover in all three classes of RNR. The functional and structural similarities suggest that the present-day RNRs have all evolved from a common ancestral reductase. Nevertheless, the different cofactors found in the three classes of RNR make the RNR proteins into interesting model systems for quite diverse protein families, such as diiron-oxygen proteins, cobalamin-dependent proteins, and SAM-dependent iron-sulfur proteins. There are also significant variations within each of the three classes of RNR. With new structures available of the R2 protein of class I RNR, we have made a comparison of the diiron centres in R2 from mouse and Escherichia coli. The R2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms, and new radical forms are different from non-radical forms. In mouse R2, the binding of iron(II) or cobalt(II) to the four metal sites shows high cooperativity. A unique situation is found in RNR from baker's yeast, which is made up of heterodimers, in contrast to homodimers, which is the normal case for class I RNR. Since the reduction of ribonucleotides is the rate-limiting step of DNA synthesis, RNR is an important target for cell growth control, and the recent finding of a p53-induced isoform of the R2 protein in mammalian cells has increased the interest for the role of RNR during the different phases of the cell cycle.
Proceedings of the National Academy of Sciences, 1997
The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B 12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90°C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B 12 . Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded ͞␣-barrel in the active site. From the conservation of substrate-and effector-binding residues we propose that the B 12 -dependent class II enzymes contain a similar barrel.
Journal of Biological Chemistry, 2001
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 ␣ 2  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. Ribonucleotide reductases provide all living organisms with the deoxyribonucleoside triphosphates required for DNA synthesis (1-3). During evolution the appearance of the first reductase was a prerequisite for the transition from an RNA world to a DNA world (4, 5). From this first enzyme, the three classes of ribonucleotide reductases of today have evolved. Enzymes belonging to different classes differ in their sensitivity to molecular oxygen; class I, present in all higher organisms and in many microorganisms, is oxygen-dependent; class III, present in microorganisms with anaerobic metabolism, is poisoned by oxygen; and class II, present in prokaryotes and a few lower
Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli
Journal of Biological Chemistry, 2004
Ribonucleotide reductase is a heterodimeric (␣ 2  2) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (؊163 ؎ 3 mV) different from that of R2 Met produced by hydroxyurea (؊115 ؎ 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming ؊223 ؎ 6 and ؊226 ؎ 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to ؊192 ؎ 2 mV for dGDP/dTTP and to ؊203 ؎ 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mM) and dATP (0.3-0.6 mM) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex. Ribonucleotide reductases (RNRs) 1 catalyze the reduction of the four canonical ribonucleotides to the corresponding de