Abstract
Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α(2) subunit that houses the active site and allosteric regulatory sites and a β(2) subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α(2)β(2) state allowing for radical transfer from β(2) to α(2) RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α(4)β(4) ring-like state. Here, we investigate whether the α(4)β(4) formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α(2), S39R-α(2), S39F-α(2), E42K-α(2), or L43Q-α(2)) that disrupt the α(4)β(4) oligomer abrogate dATP-mediated inhibition, consistent with the idea that α(4)β(4) formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-β interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-β interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α(2) or E42K-α(2)) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.