Ribonucleotide reductase in Bacillus subtilis--evidence for a Mn-dependent enzyme.
The reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, a unique step in DNA formation, is catalyzed by ribonucleotide reductase (RRase), an allosterically regulated, cell cycle-dependent enzyme. This work reports a reversible impairment of DNA formation and ribonucleotide reduction upon manganese depletion in Bacillus subtilis demonstrated through in vivo labeling with necleic acid precursors and enzyme assays with ether-permeabilized cells. No deoxyadenosylcobalamin-dependent reduction of ribonucleotides was detected in the cytosol, and the properties of a partially purified enzyme fraction, i.e., sensitivity towards EDTA and hydroxyurea (HU), indicated a metal-dependent type of RRase. The enzyme was enriched by gel filtration on Superose 12 from glycerol- or fumarate-grown cells and submitted to Q-band electron paramagnetic resonance (EPR) spectroscopy for further characterization of the metal center. A distinct Mn(II) signal was obtained in both preparations characteristic of a protein-bound mangaenese in a mononuclear metal center with axial symmetry. The intensity of this Mn signal was not affected by addition of the radical scavenger HU (10 mM) but reduced in the presence of 2.5 mM EDTA. On the basis of these results, we suggest that Bacillus subtilis has a Mn-dependent ribonucleotide reductase.
Institut für Mikrobiologie der Universität, Hannover, Germany.
S F
SF
Mohamed
O R
OR
Gvozdiak
D
D
Stallmann
U
U
Griepenburg
H
H
Follmann
G
G
Auling
D001412:Bacillus subtilis / Q000201:enzymology*; D002850:Chromatography, Gel; D004247:DNA / Q000096:biosynthesis; D004492:Edetic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006918:Hydroxyurea / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology*; D008970:Molecular Weight; D012264:Ribonucleotide Reductases / Q000378:metabolism*
D008345:Manganese; D004247:DNA; D004492:Edetic Acid; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea