The anaerobic ribonucleotide reductase from Lactococcus lactis. Interactions between the two proteins NrdD and NrdG.
Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.
Torrents
E
E
Department of Biochemistry, Medical Nobel Institute, MBB, Karolinska Institutet, SE-17177 Stockholm Sweden.
Eliasson
R
R
Wolpher
H
H
Gräslund
A
A
Reichard
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002847:Chromatography, Agarose; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007477:Ions; D013294:Lactococcus lactis / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008280:Magnetics; D008956:Models, Chemical; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D007477:Ions; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C101141:anaerobic ribonucleotide reductase; D008274:Magnesium; D011188:Potassium; D004229:Dithiothreitol