Uncoupling of Allosteric and Oligomeric Regulation in a Functional Hybrid Enzyme Constructed from Escherichia coli and Human Ribonucleotide Reductase

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-alpha of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (alpha 6 in human versus alpha 4 beta 4 in Escherichia coli, wherein beta is RNR's other subunit); however, the NTD structurally interconnects individual alpha 2 or alpha 2 and beta 2 dimeric motifs within the respective alpha 6 or alpha 4 beta 4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli-CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli-beta to form an active holocomplex. However, although the NTD is the sole physical tether to support alpha 2 and/or beta 2 associations in the dATP-bound alpha 6 or alpha 4 beta 4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.