The anaerobic (Class III) ribonucleotide reductase from Lactococcus lactis- Catalytic properties and allosteric regulation of the pure enzyme system

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase, Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in E scherichia coli, The corresponding proteins, NrdD and NrdG, were purified c lose to homogeneity, The amino acid sequences of NrdD (747 residues, 84.1 k Da) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the res pective E. coli proteins. Together, they catalyze the reduction of ribonucl eoside triphosphates to the corresponding deoxyribonucleotides in the prese nce of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, pot assium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4 Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, s imilar to the E, coli NrdD and NrdG proteins. Different from E. coli, the t wo polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated, Also the FeS cluster was easily lost from NrdG, The su bstrate specificity and overall activity of the L. lactis enzyme was regula ted according to the general rules for ribonucleotide reductases. Allosteri c effecters bound to two separate sites on NrdD, one binding dATP, dGTP, an d dTTP and the other binding dATP and ATP, The two sites showed an unusuall y high degree of cooperativity with complex interactions between effecters and a fine-tuning of their physiological effects. The results with the L. l actis class III reductase further support the concept of a common origin fo r all present day ribonucleotide reductases.