Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibucterium ammoniagenes

Abstract

The manganese-containing ribonucleotide reductase previously identified in gram-positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside-diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron-containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro.The presence of thioredoxin has been demonstrated in heat-and acid-treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12000), isoelectric point (4.3) and enzyme-activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase-thioredoxin system in these bacteria.The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603 -61 1 (19SS)l suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin-dependent and the iron-containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.Ribonucleotide reduction provides the only pathway for de novo synthesis of the deoxyribonucleotides needed for DNA replication during the S-phase of a cell cycle [I-61. Three types of ribonucleotide reductases (EC 1.17.4) have been described which differ in their coenzyme or metal requirement: (a) deoxyadenosylcobalamin-(coenzyme B12-)dependent enzymes predominate in prokaryotes [4]; (b) enzymes with a binuclear, p-0x0-bridged iron(II1) center and a tyrosine free radical which has to be generated in an oxygen-dependent reaction are found in eukaryotes, Escherichia coli and viruses [5, 61; (c) a recently identified group of manganoenzymes catalyzes ribonucleotide reduction in gram-positive bacteria of the Brevibacterium, Micrococcus and Arthrobacter genera [7 -91. These different enzymes all exhibit complex protein subunit structures and their activity is controlled by ribo-and deoxyribonucleotides, dithiols and additional metal ions in complex ways. It is likely that additional types exist, for example in strictly anaerobic archaebacteria [lo]. To assess the physiological roles of deoxyribonucleo...

The Manganese-containing Ribonucleotide Reductase ofCorynebacterium ammoniagenes Is a Class Ib Enzyme

“…The specific activity of the Mn-containing RNR of C. ammoniagenes obtained by us, even if improved at least an order of magnitude compared with previous studies (18,23), is only 12 and 18% of the specific activities described for class Ib RNR from S. typhimurium and L. lactis, respectively (10,12). There are some obvious reasons for the low enzyme activity obtained by us.…”

“…This enzyme was, however, shown to have certain features (e.g. hydroxyurea sensitivity and polypeptide sizes) in common with the well characterized class I RNR of eukaryotes and bacteria (18,23). Our purpose was to establish whether the manganese-dependent RNR really is a new class that could be fitted into the evolutionary pattern described by the other three classes.…”

“…The partially purified C. ammoniagenes RNR was suggested to consist of two subunits (18,23), a nucleotide-binding component called B1 (in this report renamed R1E) and a metalcontaining component called B2 (in this report renamed R2F). The presence of Mn was suggested on the basis of specific 54 Mn incorporation into the R2F subunit, as well as appearance of a characteristic Mn six-line EPR spectrum after denaturation of a protein preparation containing R2F.…”

Characterization of a broad-range disulfide reductase from Streptomyces clavuligerus and its possible role in beta-lactam antibiotic biosynthesis

“…Comparison of their amino acid sequences with the corresponding sequences of thioredoxin and thioredoxin reductase should establish definitively the structural relatedness of the S. clavuligerus disulfide reductase system to that of thioredoxin. Willing et al (22) reported the presence in the gram-positive Brevibacterium ammoniagenes of a thioredoxin system and showed that reduced thioredoxin was a hydrogen donor for ribonucleotide reduction. An NADPHdependent thioredoxin reductase was also detected in cell extracts, but the enzyme was not purified (22).…”

“…Willing et al (22) reported the presence in the gram-positive Brevibacterium ammoniagenes of a thioredoxin system and showed that reduced thioredoxin was a hydrogen donor for ribonucleotide reduction. An NADPHdependent thioredoxin reductase was also detected in cell extracts, but the enzyme was not purified (22). Kollarova et al (13) reported that a thioredoxin-like protein in Streptomyces aureofaciens could serve as a hydrogen donor for its own ribonucleotide reductase.…”