Back to DNA synthesis

Analysis of the results concerning the DNA synthesis in vitamin Bn-deficient cells allowed us to conclude that a vitamin Bn-independent system may operate in these cells. This system has been investigated in a series of studies (Iordan et al., 1986 Iordan, 1992 Iordan and Petukhova, 1995). After a number of passages in a medium carefully depleted of cobalt (which simulated natural habitats) the cells of P. shermanii contained less than 2 iig of cobalamins per g biomass. These cells, adapted to the vitamin B -free medium, are referred to as vitamin B -depleted cells. The synthesis and activities of ribonucleotide reductase (RNR) were compared in vitamin B -replete (Bi2 ), Bu-deficient and Bn-depleted cells. 

It was found that the DNA content in B and B -depleted cells was approximately the same, but higher than in vitamin Bn-deficient cells. Hydroxyurea, a specific inhibitor of metal-dependent RNR enzymes, suppressed the RNR activity in vitamin B -depleted cells (Fig. 5.10). In addition, the RNR activity of vitamin Bn-depleted cells was inhibited by pM concentrations of AdoCbl in the incubation mix (Fig. 5.11). Furthermore, the RNR activity of vitamin B -depleted cells showed a requirement for manganese ions (Fig. 5.12) (Iordan and Petukhova, 1995) it probably used an endogenous H-donor since it did not require dithiothreitol, a regular H-donor in B12-cells. 

The AdoCbl-independent RNR had a pH optimum at pH 7.8-8.0, while the AdoCb 1-dependent enzyme at pH 7.0. Under optimal conditions the RNR activity of Bu -cells was about 80 nmol ADP reduced per h per mg protein in the presence of 0.5 mM ADP and 30 mM DTT (reduced glutathione was an effective H-donor). Mg-ions practically were not required. In the same extracts the RNR activity with the pH optimum at 8.0 was also found (in the presence of 3 mM ADP, 30 mM DTT, 3 mM Mg-acetate), but it was twice as low (40-45 nmol ADP reduced per h per mg protein) as the activity of the AdoCbl-dependent RNR (pH optimum at 7.0). 

In vitamin Bi2-deficient cells the RNR activity at pH 8.0 was close to that of Bi2 -cells. The activity of AdoCbl-dependent RNR (pH 7.0) was undetectable in these cells. However, if AdoCbl was added to the incubation 

Recently, it has been shown (Iordan and Petukhova, 1995) that the AdoCbl-independent RNR (metal-, probably manganese-dependent) is a molecular oxygen-consuming system (cf Fig. 5.12), different from AdoCbl-dependent enzymes that function in the absence of air, but similar to mammalian RNRs that also require oxygen (Probst et al., 1989). 

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