Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets

Abstract

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs’ central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay

“…First, assume that the ratio of α:β is 1:1 and that α 2 β 2 is the active form. We believe this is a reasonable assumption based on the recent structures of an active E. coli Ia RNR 15 and structures of B. subtilis 43 and S. typhimurium 46 Ib RNRs.…”

“…α is now known to reside in many distinct quaternary structures (monomer, dimer, non-canonical dimer, tetramer, hexamer, fibrils) and the basis for these changes reside, in part in its N-terminal, ATP cone domain or partial cone domains. 15 Purification of α is often facilitated by appendage of an N-terminal (His) 6 -tag. An altered N-terminally tagged α must thus be used with caution.…”

“…Our recent model for dNDP formation in the absence of endogenous reducing system (steps A-G) and in its presence (step H) is shown in Scheme 1. 15 Many distinct perturbative biochemical studies and recent structural studies of an active α 2 β 2 double mutant (F 3 Y 122 /E 52 Q-β 2 /α 2 /GDP/TTP) establish asymmetry within the "active" complex. 15 We propose a model in which α 2, loaded with substrate (CDP) and allosteric effector (ATP), binds to β 2 and that one dCDP is formed in an α/β pair before triggering formation of a second dCDP in the adjacent α/β pair.…”

“…In the presence of endogenous reductant, we also establish that β 2 can function catalytically. However, under the conditions of limiting α 2 or at high concentrations of the subunits (1 -10 μM), the rate-limiting step switches from the conformational gating of radical transfer (RT) chemistry to re-reduction of α 2 , likely involving conformational change(s) 15 or interconversion of active and inactive quaternary structures of RNR (α 2 β 2 and α 4 β 4 in E. coli) 19 or both.…”

Ribonucleotide reductase, a novel target for gonorrhea

“…The two subunits, however, have weak affinity with the K D ~0.2 µM for Ec and human RNRs; therefore, each subunit was cloned, expressed, and purified independently. Dynamic subunit interactions are unusual for enzymes (Greene 2020), and for all RNRs the subunits are purified independently. Furthermore, assay protocols are optimized for each RNR from each organism.…”