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Related Experiment Videos

Ribonucleotide reductase in Bacillus subtilis--evidence for a Mn-dependent enzyme

S F Mohamed1, O R Gvozdiak, D Stallmann

  • 1Institut für Mikrobiologie der Universität, Hannover, Germany.

Biofactors (Oxford, England)
|July 17, 1998
PubMed
Summary

Bacillus subtilis ribonucleotide reductase (RRase) activity, essential for DNA formation, was impaired by manganese depletion. This study identifies a manganese-dependent RRase in Bacillus subtilis, crucial for DNA synthesis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Microbiology

Background:

  • Ribonucleotide reductase (RRase) catalyzes the essential conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis.
  • RRase is an allosterically regulated, cell cycle-dependent enzyme.
  • Understanding RRase in different organisms is key to comprehending DNA replication and repair.

Purpose of the Study:

  • To investigate the role of manganese in ribonucleotide reduction in Bacillus subtilis.
  • To characterize the metal cofactor dependency of Bacillus subtilis RRase.
  • To elucidate the catalytic mechanism and metal center of Bacillus subtilis RRase.

Main Methods:

  • In vivo labeling with nucleic acid precursors.
  • Enzyme assays with ether-permeabilized cells.

Related Experiment Videos

  • Enzyme purification via gel filtration and characterization using Q-band electron paramagnetic resonance (EPR) spectroscopy.
  • Sensitivity assays with EDTA and hydroxyurea (HU).

    • Main Results:

    • Manganese depletion reversibly impaired DNA formation and ribonucleotide reduction in Bacillus subtilis.
    • No deoxyadenosylcobalamin-dependent RRase activity was detected in the cytosol.
    • Partially purified RRase showed sensitivity to EDTA and HU, indicating a metal-dependent enzyme.
    • EPR spectroscopy revealed a Mn(II) signal characteristic of a mononuclear metal center with axial symmetry.
    • The Mn signal intensity was reduced by EDTA but unaffected by HU.

    Conclusions:

    • Bacillus subtilis possesses a manganese-dependent ribonucleotide reductase.
    • Manganese is essential for the activity of Bacillus subtilis RRase.
    • The enzyme likely features a mononuclear manganese center crucial for catalysis.