Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase.

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 ?2 subunit that houses the active site and allosteric regulatory sites and a ?2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact ?2?2 state allowing for radical transfer from ?2 to ?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 ? promotes formation of an ?4?4 ring-like state. Here, we investigate whether the ?4?4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the ?-? interface (S37D/S39A-?2, S39R-?2, S39F-?2, E42K-?2, or L43Q-?2) that disrupt the ?4?4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that ?4?4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the ?-? 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 ?-? interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-?2 or E42K-?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.

SUBMITTER: Chen PY 

PROVIDER: S-EPMC6028975 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature