Ribonucleotide reductases (RNRs) catalyze the de novo reduction of NDPs (CDP, UDP, ADP, GDP) to deoxynucleotides (dNDPs) in all organisms, controlling their relative ratios and amounts and contributing to the fidelity of DNA replication and repair. The class Ia RNRs are composed of alpha (a) and beta (b) subunits that form an active, dynamic a2b2 complex. Studies over the last 30 years with mechanism-based inhibitors (MBI) such the diphosphates of 2’-azido-2’-deoxycytidine and gemcitabine revealed half-sites reactivity with only one equivalent of inhibitor required per a2b2 for enzyme inactivation. Studies with unnatural amino acids (UAA) site-specifically replacing the essential Y122 radical in b2 or other residues within the RT pathway [(Y122-Y356 )-b to (Y730-Y731-C439)-a], also reveal half-sites reactivity. When 2,3,5-F3Y122 radical a hotter oxidant than Y122 radical, is incubated with a2, substrate [GDP or CDP], and effector (TTP or ATP, respectively), the conformational gating that rate-limits NDP reduction unmasks both nucleotide reduction and RT chemistry. We have recently shown that generating a radical within the RT pathway or in the active site causes formation of a tightened “a2b2” complex. These perturbations have resulted in stalled complexes of RNR which have lent themselves to structural analysis. The studies together reveal the asymmetric interaction within the active “a2b2” complex and provide insight about the dynamics of the Ia RNRs and their central role in nucleic acid metabolism . A model for the E. coli RNR structure will be proposed.

Flexibility of ribonucleotide reductases revealed in many ways

2019 Fall National Meeting

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