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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
Zip Code
Wei, Yifeng; Funk, Michael A; Rosado, Leonardo A et al. (2014) The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant. Proc Natl Acad Sci U S A 111:E3756-65
Zhang, Yan; Li, Haoran; Zhang, Caiguo et al. (2014) Conserved electron donor complex Dre2-Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis. Proc Natl Acad Sci U S A 111:E1695-704
Wei, Yifeng; Mathies, Guinevere; Yokoyama, Kenichi et al. (2014) A chemically competent thiosulfuranyl radical on the Escherichia coli class III ribonucleotide reductase. J Am Chem Soc 136:9001-13
Worsdorfer, Bigna; Conner, Denise A; Yokoyama, Kenichi et al. (2013) Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. J Am Chem Soc 135:8585-93
Minnihan, Ellen C; Nocera, Daniel G; Stubbe, Joanne (2013) Reversible, long-range radical transfer in E. coli class Ia ribonucleotide reductase. Acc Chem Res 46:2524-35
Pizano, Arturo A; Olshansky, Lisa; Holder, Patrick G et al. (2013) Modulation of Y356 photooxidation in E. coli class Ia ribonucleotide reductase by Y731 across the *2:*2 interface. J Am Chem Soc 135:13250-3
Offenbacher, Adam R; Minnihan, Ellen C; Stubbe, JoAnne et al. (2013) Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase ýý2. J Am Chem Soc 135:6380-3
Zhang, Yan; An, Xiuxiang; Stubbe, Joanne et al. (2013) Investigation of in vivo roles of the C-terminal tails of the small subunit (ýýýý') of Saccharomyces cerevisiae ribonucleotide reductase: contribution to cofactor formation and intersubunit association within the active holoenzyme. J Biol Chem 288:13951-9
Ando, Nozomi; Brignole, Edward J; Zimanyi, Christina M et al. (2011) Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase. Proc Natl Acad Sci U S A 108:21046-51
Minnihan, Ellen C; Young, Douglas D; Schultz, Peter G et al. (2011) Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase. J Am Chem Soc 133:15942-5

Showing the most recent 10 out of 95 publications