Deoxyribonucleotide synthesis in anaerobic microorganisms: The class III ribonucleotide reductase
For growth under oxygen-free atmosphere, some strict or facultative anaerobes depend on a class III ribonucleotide reductase for the synthesis of deoxyribonucleotides, the DNA precursors. Prototypes for this class of enzymes are ribonucleotide reductases from Escherichia coli and bacteriophage T4. This review article describes their structural and mechanistic properties as well as their complex allosteric regulation. Their evolutionnary relationship to class I and class II ribonucleotide reductases is also discussed.
Available from: Eric Pilet
- "HydG was previously shown to reductively cleave AdoMet into 5 0 -deoxyadenosine (AdoH) and methionine in the absence of substrate , provided it is anaerobically incubated with dithionite as a reducing agent . Substrate-independent AdoMet cleavage is common within Radical-SAM enzymes, as shown in the case of Biotin Synthase , the activating component of ribonucleotide reductase  or the spore photoproduct lyase . "
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ABSTRACT: [FeFe]-hydrogenases catalyze the protons/hydrogen interconversion through a unique di-iron active site consisting of three CO and two CN ligands, and a non-protein SCH(2)XCH(2)S (X=N or O) dithiolate bridge. Site assembly requires two "Radical-S-adenosylmethionine (SAM or AdoMet)" iron-sulfur enzymes, HydE and HydG, and one GTPase, HydF. The sequence homology between HydG and ThiH, a Radical-SAM enzyme which cleaves tyrosine into p-cresol and dehydroglycine, and the finding of a similar cleavage reaction catalyzed by HydG suggests a mechanism for hydrogenase maturation. Here we propose that HydG is specifically involved in the synthesis of the dithiolate ligand, with two tyrosine-derived dehydroglycines as precursors along with an [FeS] cluster of HydG functioning both as electron shuttle and source of the sulfur atoms.
Available from: Marc Zapatka
- "Furthermore, two reactions, i.e. for the anaerobic nucleoside-triphosphate reductase activating system and the component ribonucleoside triphosphate reductase activase were up-regulated. These reactions needed to synthesise deoxyribonucleotides under anaerobic conditions [42,43] are showing expression patterns analogous to pyruvate formate-lyase activase . Also up-regulated was the anaerobic coproporphyrinogen III oxidase. "
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ABSTRACT: Biochemical investigations over the last decades have elucidated an increasingly complete image of the cellular metabolism. To derive a systems view for the regulation of the metabolism when cells adapt to environmental changes, whole genome gene expression profiles can be analysed. Moreover, utilising a network topology based on gene relationships may facilitate interpreting this vast amount of information, and extracting significant patterns within the networks.
Interpreting expression levels as pixels with grey value intensities and network topology as relationships between pixels, allows for an image-like representation of cellular metabolism. While the topology of a regular image is a lattice grid, biological networks demonstrate scale-free architecture and thus advanced image processing methods such as wavelet transforms cannot directly be applied. In the study reported here, one-dimensional enzyme-enzyme pairs were tracked to reveal sub-graphs of a biological interaction network which showed significant adaptations to a changing environment. As a case study, the response of the hetero-fermentative bacterium E. coli to oxygen deprivation was investigated. With our novel method, we detected, as expected, an up-regulation in the pathways of hexose nutrients up-take and metabolism and formate fermentation. Furthermore, our approach revealed a down-regulation in iron processing as well as the up-regulation of the histidine biosynthesis pathway. The latter may reflect an adaptive response of E. coli against an increasingly acidic environment due to the excretion of acidic products during anaerobic growth in a batch culture.
Based on microarray expression profiling data of prokaryotic cells exposed to fundamental treatment changes, our novel technique proved to extract system changes for a rather broad spectrum of the biochemical network.
Available from: oregonstate.edu
- "Besides aerobic ribonucleotide reductase (abbreviated as RNR), both E. coli and T4 also encode a Class III ribonucleotide reductase, the anaerobic form (Young et al., 1994a; Olcott et al., 1998; Fontecave et al., 2002). "
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ABSTRACT: The enzymes of dNTP synthesis in T4 infection associate to form a multienzyme complex, the T4 dNTP synthetase complex, facilitating the flow of metabolites en route to dNTPs, and their subsequent flow into DNA. Study of protein-protein interactions helps one to understand how the enzymes in the complex are organized and coordinated to function efficiently. By use of several approaches, namely, IAsys optical biosensing, fluorescence spectroscopy and analytical ultracentrifugation, a large number of direct protein-protein interactions among the proteins in the complex were detected. Those associations involved not only T4-encoded proteins but also two host-encoded enzymes, namely, E. coli NDP kinase and adenylate kinase. Quantitative analysis of some of those associations show that their equilibrium dissociation constants fall into the micromolar range, which is comparable to the estimated intracellular protein concentrations, suggesting that those interactions are significant in vivo. In addition, direct interactions between T4 single-strand DNA binding protein (gp32) and several proteins in the complex suggest a linkage between the dNTP synthetase complex and DNA replisome. We also found that some nucleotides, especially ATP, enhanced most of the direct protein-protein interactions. Quantitative analysis shows that, in the presence of 1 mM ATP, the dissociation constants were an order of magnitude lower than that in the absence of ATP. The intracellular concentration of ATP was determined in millimolar range, suggesting that in vivo the associations are even more significant. IAsys analysis shows the self-association of E. coli NDP kinase and its enhancement by ATP. Equilibrium sedimentation indicates that, in the absence of ATP, the dissociation constant between dimers and tetramers was 0.8 uM. However, in the presence of 0.5 mM ATP, NDP kinase appeared completely in tetramer, suggesting that ATP might exert its effect through influence upon the quaternary structure of NDP kinase. A mixture of purified T4 enzymes was assayed for activity of a three-step sequence (dCTP->dCMP->dUMP->dTMP), sequentially catalyzed by dCTPase/dUTPase, dCMP deaminase and thymidylate synthase. Kinetic coupling behavior was observed. Other proteins in the complex that are not catalytically involved in this pathway enhanced the kinetic coupling, suggesting positive cooperativity among interactions stabilizing the complex. Printout. Thesis (Ph. D.)--Oregon State University, 2007. Includes bibliographical references (leaves 160-180).
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