Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and are responsible for the supply of monomeric precursors required for DNA replication and DNA repair. The central role of RNRs in nucleic acid metabolism has made them the successful target of at least three drugs used clinically in the treatment of cancers, hydroxyurea, gemcitabine, and clofarabine, as well as the clinical drug candidate, triapine. The RNRs serve as a paradigm for understanding the exquisite control of reactions involving stable and transient protein- and nucleotide-based radical intermediates. The class Ia enzymes also serve as the paradigm for an unprecedented mechanism of radical propagation over a distance of >35 ?, involving the transient formation of aromatic amino acid radical intermediates (Y or W) by proton-coupled electron transfer (PCET). The present proposal is focused on the class Ia RNRs from E. coli and human. Our ability to incorporate unnatural amino acids (UAAs) site-specifically into the two subunits (? and ?) of the E. coli RNR, coupled with the tools of high field EPR and ENDOR spectroscopies, stopped-flow kinetics, structure, and computation, will allow us to determine if the PCET process occurs with proton transfer that is orthogonal or co-linear to the proposed ET pathway. The UAAs 3-nitrotyrosine (NO2Y) and 3- aminotyrosine (NH2Y) serve as probes of the ground state and intermediate states, respectively, of E. coli RNR, and are sensitive to changes in protein conformation, hydrogen bonding networks, and redox potentials. A method for the site-specific incorporation of the UAA 2,3,5-trifluorotyrosine (F3Y) into ? will be developed, and the pH rate profile of the resulting mutant-?s will be measured to test the proposed differences in PCET mechanism between the two subunits. Humans have two RNRs: ?/?, involved in DNA replication, and ?/?', involved in DNA repair and mitochondrial DNA replication. Basic biochemical studies of these two RNRs will be carried out to understand (a) the stability of the radical initiation cofactor, (b) the ability of ?'to function as a catalase, (c) the active quaternary structures of ?/? and ?/?', and (d) the similarities or differences in the PCET pathway compared to the E. coli enzyme.
Ribonucleotide reductases (RNRs) are enzymes responsible for the conversion of nucleotides to deoxynucleotides, the monomeric building blocks of DNA. Thus, they play a key role in regulating DNA replication and repair, and are a successful clinical target for several types of cancer.
The aim of this proposal is to study RNR's unique mechanism, the understanding of which will aid in developing novel RNR inhibitors.
|Ravichandran, Kanchana; Minnihan, Ellen C; Lin, Qinghui et al. (2017) Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry 56:856-868|
|Lin, Qinghui; Parker, Mackenzie J; Taguchi, Alexander T et al. (2017) Glutamate 52-? at the ?/? subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer. J Biol Chem 292:9229-9239|
|Greene, Brandon L; Taguchi, Alexander T; Stubbe, JoAnne et al. (2017) Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase. J Am Chem Soc 139:16657-16665|
|Ravichandran, Kanchana R; Zong, Allan B; Taguchi, Alexander T et al. (2017) Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer. J Am Chem Soc 139:2994-3004|
|Nick, Thomas U; Ravichandran, Kanchana R; Stubbe, JoAnne et al. (2017) Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase. Biochemistry 56:3647-3656|
|Olshansky, Lisa; Greene, Brandon L; Finkbeiner, Chelsea et al. (2016) Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase. Biochemistry 55:3234-40|
|Olshansky, Lisa; Stubbe, JoAnne; Nocera, Daniel G (2016) Charge-Transfer Dynamics at the ?/? Subunit Interface of a Photochemical Ribonucleotide Reductase. J Am Chem Soc 138:1196-205|
|Oyala, Paul H; Ravichandran, Kanchana R; Funk, Michael A et al. (2016) Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes: E. coli Ribonucleotide Reductase As an Example. J Am Chem Soc 138:7951-64|
|Ando, Nozomi; Li, Haoran; Brignole, Edward J et al. (2016) Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer. Biochemistry 55:373-81|
|Ravichandran, Kanchana R; Taguchi, Alexander T; Wei, Yifeng et al. (2016) A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes. J Am Chem Soc 138:13706-13716|
Showing the most recent 10 out of 121 publications