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 #
5R01GM029595-35
Application #
8516043
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
2013-08-01
Budget End
2014-07-31
Support Year
35
Fiscal Year
2013
Total Cost
$449,036
Indirect Cost
$167,059
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
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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
Olshansky, Lisa; Stubbe, JoAnne; Nocera, Daniel G (2016) Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase. J Am Chem Soc 138:1196-205
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
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Ravichandran, Kanchana R; Minnihan, Ellen C; Wei, Yifeng et al. (2015) Reverse Electron Transfer Completes the Catalytic Cycle in a 2,3,5-Trifluorotyrosine-Substituted Ribonucleotide Reductase. J Am Chem Soc 137:14387-95
Wei, Yifeng; Li, Bin; Prakash, Divya et al. (2015) A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase. Biochemistry 54:7019-28
Song, David Y; Pizano, Arturo A; Holder, Patrick G et al. (2015) Direct Interfacial Y731 Oxidation in α2 by a Photoβ2 Subunit of E. coli Class Ia Ribonucleotide Reductase. Chem Sci 6:4519-4524
Nick, Thomas U; Lee, Wankyu; Kossmann, Simone et al. (2015) Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase. J Am Chem Soc 137:289-98
Doan, Peter E; Shanmugam, Muralidharan; Stubbe, JoAnne et al. (2015) Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase. J Am Chem Soc 137:15558-66

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