Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAEcellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 ( M , =z 80000) and catalytic protein B2 (Mr = 100000, composed of two M , = 50000 polypeptides), which were both necessary for activity.In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (Z, o = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(II1)-containing pseudocatalase and of 0x0-bridged binuclear Mn(ll1) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(1l) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity.Manganese-deficient cell cultures were also grown in the presence of 54MnC12. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled.All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.

A gene has been cloned from Trypanosoma brucei which encodes a protein of 144 amino acid residues containing the thioredoxin-like motif WCPPCR. Overexpression of the gene in E. coli resulted in 4 mg pure protein from 100 ml bacterial cell culture. Recombinant T. brucei tryparedoxin acts as a thiol-disulfide oxidoreductase. It is spontaneously reduced by trypanothione. This dithiol, exclusively found in parasitic protozoa, also reduces E. coli glutaredoxin but not thioredoxin. The trypanothione/tryparedoxin couple is an effective reductant of T. brucei ribonucleotide reductase. Like thioredoxins it has a poor GSH:disulfide transhydrogenase activity. The catalytic properties of tryparedoxin are intermediate between those of classical thioredoxins and glutaredoxins which indicates that these parasite proteins may form a new class of thiol-disulfide oxidoreductases.z 1998 Federation of European Biochemical Societies.

Reductive elimination of the 2'-hydroxyl group from ribonucleotides to yield 2'-deoxyribonucleotides, the monomeric precursors of DNA, requires an uncommon type of enzyme catalysis in which the transition metals, manganese, iron, or cobalt, and free radical intermediates cooperate. In the group of deoxyadenosylcobalamin (coenzyme B 12)-dependent ribonucleotide reductases the coenzyme supplies a transient radical pair of deoxyadenosyl, and cob(II)alamin whereas in the nonheme-iron group of enzymes a protein subunit carries a stable tyrosyl radical coordinated to a binuclear iron(III) complex; the manganese-dependent enzymes are less precisely known. The radicals are thought to function in hydrogen transfer from cysteine SH to the ribonucleotide substrate, and the metal complexes are apparently needed to generate and stabilize the radicals. In aerobic organisms oxygen also plays a critical role in these processes and hence in DNA synthesis and cell proliferation. In addition to the transition metals, Mg 2+ or Ca 2+ are required by several ribonucleotide reductases for structural integrity. However the most potent inhibitors of deoxyribonucleotide biosynthesis (of potential interest in chemotherapy) are not metal chelators but radical scavengers. Cell cycle arrest and cell death produced by simple chemicals like N-hydroxyurea and hydroxamates can be traced back to their reaction with ribonucleotide reductase. Evidence is accumulating that independent enzymes of deoxyribonucleotide and DNA synthesis are functionally coupled in a novel type of supramolecular structure.

All known cosmic and geological conditions and laws of chemistry and thermodynamics allow that complex organic matter could have formed spontaneously on pristine planet Earth about 4,000 mya. Simple gasses and minerals on the surface and in oceans of the early Earth reacted and were eventually organized in supramolecular aggregates and enveloped cells that evolved into primitive forms of life. Chemical evolution, which preceded all species of extant organisms, is a fact. In this review, we have concentrated on experimental and theoretical research published over the last two decades, which has added a wealth of new details and helped to close gaps in our previous understanding of this multifaceted field. Recent exciting progress in the molecular and genetic analyses of existing life, in particular microorganisms of ancient origin, even supports the possibility that a cellular, self-reproducing common ancestor might be assembled and resurrected in anaerobic cultures at some time in the future. Charles Darwin did not, and indeed, could not, address and specify the earliest phases of life which preceded the Origin of Species. However, in a famous letter, he sketched "a warm little pond with all sorts of... (chemicals, in which) ...a protein was chemically formed." We try to trace the impact of his charming clear-sighted metaphor up to the present time.

In many physiological studies dehydroascorbate (DHA) reductase is regarded as one of the chloroplast enzymes involved in the protection against oxidative stress. Here, evidence is presented that plant cells do not possess a specific DHA reductase. The DHA reductase activities measured in plant extracts are due to side reactions of proteins containing redoxactive dicysteine sites. Native gel electrophoresis combined with specific activity staining revealed three different proteins with DHA reductase activity in leaf and chloroplast extracts. These proteins have been identified as thioredoxins and trypsin inhibitors (Kunitz type) by Western blot analysis. The essential regulatory functions of thioredoxins in chloroplast metabolism are strongly inhibited in the presence of as little as 50 μΜ DHA. Thus, the intracellular DHA concentration should be kept below 50 μΜ but not all proteins with DHA reductase activity are effective enough for this purpose. A specific DHA reductase is frequently demanded as part of the enzymatic equipment to avoid oxidative stress. We argue that this is not necessary because in chloroplasts DHA does not accumulate to any significant extent due to the high activities of monodehydroascorbate reductase and of reduced ferredoxin.