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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029595-32
Application #
7980636
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1987-09-01
Project End
2014-07-31
Budget Start
2010-09-15
Budget End
2011-07-31
Support Year
32
Fiscal Year
2010
Total Cost
$482,382
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Greene, Brandon L; Stubbe, JoAnne; Nocera, Daniel G (2018) Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase. J Am Chem Soc 140:15744-15752
Brignole, Edward J; Tsai, Kuang-Lei; Chittuluru, Johnathan et al. (2018) 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound. Elife 7:
Lee, Wankyu; Kasanmascheff, Müge; Huynh, Michael et al. (2018) Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm. Biochemistry 57:3402-3415
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
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
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
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
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
Kasanmascheff, Müge; Lee, Wankyu; Nick, Thomas U et al. (2016) Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-? mutant unmasks a new conformation of the pathway residue 731. Chem Sci 7:2170-2178

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