Ribonucleotide reductase in cell extracts of Methanobacterium thermoautotrophicum

Escherichia coli obtains the deoxyribonucleotides required for DNA synthesis from ribonucleotides through the activity of the enzyme ribonucleoside diphosphate reductase (1-3). A closely related enzyme has the same function in mammalian cells. Both enzymes consist of two nonidentical subunits, proteins B1 and B2 (4) of E. coli and the corresponding mammalian proteins Ml and M2 (5). B2 and M2 contain a tyrosyl radical (6-8) that has arisen by oxidation of a specific tyrosine residue of the polypeptide chain. An oxygenrequiring enzyme system catalyzes the formation of this radical in E. coli (9, 10). However, E. coli grows well also under anaerobic conditions. This poses the question how deoxyribonucleotides are synthesized in the absence of oxygen. One could imagine that during anaerobiosis an electron acceptor other than oxygen might function in radical generation. Or, deoxyribonucleotides might be formed by other enzymes. The second alternative was favored strongly from genetic experiments (11-13) demonstrating that mutations in the nrdA or nrdB gene, coding for protein B1 or B2, did not affect the anaerobic growth of the bacteria. These experiments did, however, not distinguish between a different ribonucleotide reductase or a completely different mode of synthesis-e.g., via the deoxyribose 5-phosphate aldolase pathway (15).Here we first establish from isotope experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. We then describe the existence of a different reductase in extracts from anaerobically grown bacteria. The enzyme is not inhibited by antibodies raised against protein B1 or B2 and is only moderately sensitive to hydroxyurea, a drug that strongly inhibits the aerobic reductase, but is rapidly inactivated by air. The substrate of the reaction appears to be CTP and not CDP, as for the aerobic reaction. MATERIALS AND METHODSBacterial Strains. The E. coli K-12 strain AB1157 and its oxygen-sensitive derivative Oxys-11 (11) were obtained from H. I. Adler (Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN). E. coli So1452, a strain with an insertion mutation in the gene for cytidine deaminase, was a gift from B. Mygind (Institute for Biological Chemistry, University of Copenhagen, Denmark).Preparation of Bacterial Extracts. Five-liter cultures of AB1157 or Oxys-11 were grown anaerobically in Luria broth containing 1 g of glucose per liter under continuous bubbling with 4% C02/96% N2 in a Microferm New Brunswick fermentor with constant pH control. When the bacteria had reached midlogarithmic phase (OD = 1.0 at 640 nm) they were harvested by centrifugation in twelve 0.5-liter bottles in cold Sorvall centrifuges, resuspended under a flow of argon in a small volume of an anaerobic 50 mM Tris buffer (pH 7.5), and transferred under argon to four preweighed capped Beckman no. 355603 thick-walled polycarbonate tubes. After centrifugation and removal of the supernatant solution, the weighed bacteria (1-2 g per tube) were stored under liquid...

Section: Discussionmentioning

confidence: 99%

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Fontecave

Eliasson

Reichard

1989

“…(fix) However, three (and possibly four) mechanistically different RNRs are known in the modern world (34,35). This implies that the RNRs in different kingdoms are not homologous, and makes it impossible to assign by parsimony a chemical reaction mechanism to any RNR presumed to have been present in the progenote.h Indeed, in the absence of evidence for points i and ii, it would be reasonable to suggest that the progenote did not contain any RNR and that the mechanistically different RNRs in the modern world reflect the independent origin of several types of RNR after the divergence of the progenote.…”

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confidence: 99%

Modern metabolism as a palimpsest of the RNA world.

Benner

Ellington

Tauer

1989

429

324

An approach is developed for constructing models of ancient organisms using data from metabolic pathways, genetic organization, chemical structure, and enzymatic reaction mechanisms found in contemporary organisms. This approach is illustrated by a partial reconstruction of a model for the "breakthrough organism," the last organism to use RNA as the sole genetically encoded biological catalyst. As reconstructed here, this organism had a complex metabolism that included dehydrogenations, transmethylations, carboncarbon bond-forming reactions, and an energy metabolism based on phosphate esters. Furthermore, the breakthrough organism probably used DNA to store genetic information, biosynthesized porphyrins, and used terpenes as its major lipid component. This model differs significandy from prevailing models based primarily on genetic data.Since the discovery of self-splicing RNA (1), molecular biology has become the central focus of speculation concerning early forms of life. Many of these speculations consider genetic structure to the exclusion of most other biochemical data in modeling the "RNA world" (2-5). As discussed elsewhere, this narrow focus leads to interesting but often chemically and biologically implausible models (6-10).We develop here an alternative approach for generating experimentally testable models of the RNA world, based on metabolic, structural, and mechanistic data from contemporary organisms. Several specific "paradigms," problem solutions covering individual topics in biochemical evolution, are constructed by this approach. These paradigms demonstrate the utility of this broader view and provide elements of a framework for interpreting the "historical" component of modern biochemistry (11).We begin by assuming that life on earth passed through three episodes ( Fig. 1) (12). In the first (the RNA world) (13), RNA was the only genetically encoded component of biological catalysts. The second episode began with the invention of translation-based synthesis of proteins in a "breakthrough organism," the first organism to contain a genetically encoded messenger RNA that directed the synthesis of a protein selectable for its catalytic activity. The third episode comprises the divergent evolution of the "progenote," the most recent common ancestor of all modern forms of life.a This model views modern macromolecular catalysis as a "palimpsest" of an earlier metabolic state, with features that arose recently ("derived traits") superimposed upon features that are remnants of ancient life ("primitive traits"). (A palimpsest is a parchment that has been inscribed two or more times, with the previous texts imperfectly erased and therefore still partially legible.) To describe the biochemistry of these ancient organisms, we must first examine contemporary biochemical traits to distinguish ancient information from information added later. These descriptions are prerequisites for descriptions ofthe development ofmetabolism, the origin of translation, and other events that occurred in the RNA world....

“…Extracts from an oxygen-sensitive mutant of E. coli, lacking normal class I reductase activity, catalyzed an oxygen-sensitive reduction of ribonucleotides, different from the reaction catalyzed by the known aerobic E. coli reductase (18). A similar activity was found in extracts from the strict anaerobic Methanobacterium thermoautotrophicum (17). We now describe fractionation and initial characterization of the ribonucleotide reductase system from anaerobic E. coli.…”

mentioning

confidence: 77%

“…Fig. 4 (17). Class IV enzymes need not be restricted to prokaryotes, nor need all After 20 min, the reactions were stopped by addition of 0.3 ml of 1 M perchloric acid.…”

Section: Methodsmentioning

confidence: 99%

“…Recent genetic (15,16) and biochemical (17,18) evidence pointed to the possible existence of a fourth class, present in microorganisms during anaerobic growth. Extracts from an oxygen-sensitive mutant of E. coli, lacking normal class I reductase activity, catalyzed an oxygen-sensitive reduction of ribonucleotides, different from the reaction catalyzed by the known aerobic E. coli reductase (18).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

Eliasson

Fontecave²,

Jörnvall³

et al. 1990

Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2e ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several dermed protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein Bi) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.Ribonucleotide reductases catalyze the synthesis of the four deoxyribonucleoside triphosphates (dNTPs) required for DNA replication (1-4). The enzymes provide a link between RNA and DNA metabolism. They have attained special biological interest in connection with recent theories concerning the evolution of life on earth since their appearance was a prerequisite for the transition of an "RNA world" into a "DNA world" (5). Several forms of the enzyme occur in nature, with different structures and catalytic mechanisms. Also, their chemistry is of considerable interest. The enzyme from Escherichia coli provided the first example of a biological process that uses a protein radical as a mechanistic option (6, 7). While the direct reduction of ribonucleotides via a radical mechanism is a general mode of dNPT synthesis in all organisms, the way in which this is achieved varies. At present, three classes of ribonucleotide reductases have been described: The aerobic E. coli enzyme (8) is the prototype for class I enzymes (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), also present in all higher animal (9) and plant cells (10). These enzymes consist of two proteins: one large homodimer (protein B1 of E. coli) and one small homodimer (protein B2). Characteristic features are the presence of a ferric:tyrosyl radical center in B2 (6) and redox-active dithiols in B1 (11), both involved in the catalytic process. B1 also provides the reductase with allosteric pro...