B12-DEPENDENT RIBONUCLEOTIDE REDUCTASES FROM DEEPLY ROOTED EUBACTERIAARE STRUCTURALLY RELATED TO THE AEROBIC ENZYME FROM ESCHERICHIA-COLI

The ribonucleotide reductases from three ancient eubacteria, the hyper thermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aur antiacus, were found to be coenzyme-B-12 (class II) enzymes, similar t o the earlier described reductases from the archaebacteria Thermoplasm a acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a posit ive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR gen es were cloned by PCR from peptide sequence information. The TM gene w as 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 B-12. Structure determinations of the E. coli reductase complexed wit h substrate and allosteric effecters earlier demonstrated a 10-strande d beta/alpha-barrel in the active site. From the conservation of subst rate-and effector-binding residues we propose that the B-12-dependent class II enzymes contain a similar barrel.