Ribonucleotide reductase from Escherichia coli: Mechanistic studies of hydroxyurea resistance

Ribonucleotide reductase (RNR) is the enzyme that catalyzes the removal of the 2′-hydroxyl of ribonucleoside diphosphates, generating deoxyribonucleosides for use in DNA synthesis. As a key step in DNA replication, ribonucleotide reductase provides an attractive target for anticancer and antiviral therapies.; The purpose of this work was to create Escherichia coli ribonucleotide reductases that are resistant to the inhibitor hydroxyurea. These were characterized to obtain information on the mechanism of inhibition of ribonucleotide reductase by radical scavengers. Ultimately these mutants may prove useful in gene therapy for their protection against the myelosuppressive effects of hydroxyurea in cancer and HIV patients.; Using the method of random oligonucleotide mutagenesis coupled with genetic selection in E. coli, I have created a family of mutant R2 subunits resistant to hydroxyurea and characterized a number of these in vitro. The majority of mutants in this library conferring hydroxyurea resistance contained a single S75T mutation. Further analysis of all substitutions at Ser75 revealed that a number of Ser75 mutants confer protection in this assay, although S75T was the only substitution conferring a high level of resistance.; To define the role of Ser75 in radical stability, purified S75T R2 was compared to wild-type R2 with respect to reconstitution profile, half-life, and enzyme activity. Although 1000-fold resistant to hydroxyurea in the survival assay, S75T R2 displays only a 50% increase in half-life over wild-type protein in vitro. Furthermore, its activity is 2.6-fold lower than that of wild-type R2. Possible reasons for this disparity are discussed.; Inactive S75A R2 and moderately protective S75N R2 were also purified and examined with respect to reconstitution profiles and enzyme activity. S75A R2 fails to reconstitute and displays no detectable activity; S75N R2 partially reconstitutes, but the radical is destabilized, even in the absence of scavengers. These data indicate that Ser75 is involved in radical formation, radical stability, and enzyme activity.