The purification and characterization of an adenosylcobalamin-dependent ribonucleoside diphosphate reductase from Corynebacterium nephridii.
A deoxyadenosylcobalamin-dependent ribonucleoside diphosphate reductase has been purified to homogeneity from cell free extracts of Corynebacterium nephridii. Ion exchange chromatography of th extract on DEAE-Sephadex and DEAE-Bio-Gel A, followed by affinity chromatography on dGTP-Sepharose, yielded two forms of the reductase. The first reductase, which was weakly bound to the affinity column, was eluted with 0.1 M citrate buffer, pH 6.5, while the second, more tightly bound form required 2 M urea for its elution. The weakly bound form is homogeneous as judged by gel filtration, equilibrium sedimentation, and polyacrylamide gel electrophoresis. This enzyme is a dimeric protein with a molecular weight of 196,000, composed of two identical or very similar subunits with molecular weights of 100,000. The second protein appears to be a polymeric form of the reductase. Both forms of the enzyme catalyze the reduction of the four common ribonucleoside diphosphates as well as the hydrogen exchange between adenosylcobalamin and the solvent. The ribonucleoside diphosphate reductase system of C. nephridii appears to be intermediate between the more "primitive" adenosylcobalamin-dependent reductase system of Lactobacillus leichmannii and the more "advanced" nonheme iron system of Escherichia coli.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003038:Cobamides / Q000494:pharmacology*; D003352:Corynebacterium / Q000201:enzymology*; D003854:Deoxyribonucleotides; D007700:Kinetics; D008970:Molecular Weight; D012262:Ribonucleoside Diphosphate Reductase / Q000302:isolation & purification / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D003038:Cobamides; D003854:Deoxyribonucleotides; D012264:Ribonucleotide Reductases; D012262:Ribonucleoside Diphosphate Reductase