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 Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two 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 substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.
Torrents
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E
Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institutet, S-17177 Stockholm, Sweden.
Buist
G
G
Liu
A
A
Eliasson
R
R
Kok
J
J
Gibert
I
I
Gräslund
A
A
Reichard
P
P
D000494:Allosteric Regulation; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D009876:Operon; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D009711:Nucleotides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases