B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli (deoxyribonucleotide synthesisyallosteric regulationyevolutionythermophilic)

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-B12 (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 B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded bya-barrel in the active site. From the conservation of substrateand effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel. All living cells produce the four deoxyribonucleotides required for DNA replication and repair by reduction of ribonucleotides. Three classes of ribonucleotide reductases exist (1, 2). All use free radical chemistry for catalysis but differ in the way in which they produce a protein radical required for the activation of the substrate. Class I enzymes, with the aerobic Escherichia coli enzyme as prototype, are a2b2 proteins. The large a protein (R1) harbors catalytic and allosteric sites, whereas b contains a diferric center and a stable tyrosyl radical. They occur in eukaryotes and some aerobic eubacteria. Class II enzymes, with the Lactobacillus leichmannii enzyme as prototype, have an a or a2 structure and use adenosylcobalamin as radical generator. They occur in aerobic and anaerobic bacteria. Class III enzymes finally, with the anaerobic E. coli enzyme as prototype, have an a2b2 structure and use Sadenosylmethionine to generate a stable glycyl radical. They occur in anaerobic bacteria. The prototypes for the three classes were not homologous and it seemed possible that they had arisen separately during evolution (3). However, functional aspects are in favor of a common root (1, 2). Members of the three classes reduce ribonucleotides by an identical mechanism and critical, functionally involved cysteine residues are found in corresponding positions in the class I and II enzymes (4, 5). Furthermore the allosteric regulation of the substrate specificity of the enzymes is highly similar, suggesting that the different classes may in part have closely related tertiary structures in spite of the large divergence of their primary structures (1, 2). For a long time the only characterized class II enzyme was the prototype from L. leichmannii, a eubacterium (6, 7). However, two recent studies reported the sequences and some properties of class II reductases from two archaebacteria, Pyrococcus furiosus (8) and Thermoplasma acidophilum (9). Both sequences show considerable homology not only to the L. leichmannii enzyme but also to class I reductases. In addition, at the N terminus '100 amino acids were homologous to the corresponding sequence of the E. coli class III reductase. This result is persuasive evidence that all three classes originated from a common ancestor. Do the negative results with the Lactobacillus enzyme mean that its sequence has evolved so far that a common origin no longer is recognized? Knowledge of eubacterial class II ribonucleotide reductases is limited. We have now studied class II enzymes from some deeply rooted eubacteria (10, 11) and find that their sequences show a greater kinship to the enzymes from archaebacteria than to that from L. leichmannii. From a combination of sequence data of the class II enzymes with recent structural results from complexes between the E. coli class I reductase and allosteric effectors (12) we hypothethize that the two classes share an appreciable part of the tertiary structure involved in effector binding. To facilitate the presentation and discussion of our work we suggest the name nrdJ for the gene of class II ribonucleotide reductases. MATERIALS AND METHODS Materials. Thermotoga maritima (TM) strain MSB8 (DSM 3109 from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) and Deinococcus radiodurans R1 (ATCC 13939) (DR) were used for enzyme purification and PCR amplification of genomic DNA. Extracts from Chloroflexus aurantiacus J-10-fl (ATCC 29366), were used for enzyme assays. E. coli DH5aF9, and plasmids pBlueScript SK (1) (pBSK, Stratagene), pGEM-T (Promega) and pET22b (Novagen) were used for recombinant DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9413487-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: TM, Thermotoga maritima; DR, Deinococcus radiodurans; TIGR, The Institute for Genomic Research. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. Y12877). ¶To whom reprint requests should be addressed.