Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides in all organisms supplying the monomeric precursors required for DNA replication and repair. The class Ia and Ib RNRs are composed of two subunits (? and ?) which form the active (?2)n?2 complex. ?2 houses the essential dinuclear-tyrosyl radical (Y?) cofactor which in the class Ia RNRs from E. coli, S. cerevisiae, and humans is a diferric cluster (FeIII2-Y?), and in the class Ib RNRs from E. coli, B. subtilis, S. sanguinis and many pathogenic microorganisms is likely, a dimanganese cluster (MnIII2- Y?). We are interested in how these cofactors are biosynthesized and repaired, i.e., if the Y?is reduced inside the cell by endogenous reductants or the chemotherapeutic hydroxyurea, how the MeIII2-cluster is re-converted to active cofactor. Studies of self-assembly of FeIII2-Y?in the Ia RNRs in vitro have demonstrated the requirement for reducing equivalents, and controlled metal and oxidant delivery. We have recently discovered in E. coli, a 2Fe2S cluster ferredoxin (YfaE) that plays a role in electron transfer in cluster biosynthesis and maintenance and likely is involved in cluster metallation. In assembly of the eukaryotic class Ia cluster in S. cerevisiae, Dre2 and Tah18 are proposed to play a similar role in electron transfer for biosynthesis and maintenance and Grx3/Grx4 and Dre2/Tah18 are proposed to play a role in metallation. While the class Ib RNRs can also self-assemble FeIII2-Y?active in nucleotide reduction, our recent studies suggest that the active cofactor in vivo is MnIII2-Y?cluster and that its assembly in vitro and in vivo require an unusual flavodoxin, NrdI. The present proposal is focused on obtaining evidence for the roles of the protein factors in vivo (E. coli and S. cerevisiae) using the tools we have developed over the last few years (whole cell EPR, antibodies, activity assays, isogenic strains with gene deletions and conditional expression and X-ray crystallography). We are also investigating the mechanism of MnIII2-Y?cofactor assembly using time resolved biophysical methods (stopped flow, rapid freeze quench EPR and EXAFS spectroscopies). The observation of the long ago postulated, but elusive, Mn-RNR raises the importance of the competition between pathogens and their hosts for controlled metallation and suggests new targets for antibacterial therapeutics.

Public Health Relevance

Ribonucleotide reductases (RNRs) play an essential role in DNA replication and repair and are the target of three antitumor agents used clinically. The essential diiron and dimanganese metallo-cofactors, the former in humans and the latter in pathogenic organisms, and the discovery of unique biosynthetic machinery for their assembly, offer an additional target for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081393-06
Application #
8605195
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2008-07-02
Project End
2016-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
6
Fiscal Year
2014
Total Cost
$465,167
Indirect Cost
$111,857
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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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
Huang, Mingxia; Parker, Mackenzie J; Stubbe, JoAnne (2014) Choosing the right metal: case studies of class I ribonucleotide reductases. J Biol Chem 289:28104-11
Parker, Mackenzie J; Zhu, Xuling; Stubbe, JoAnne (2014) Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions. Biochemistry 53:766-76
Rhodes, DeLacy V; Crump, Katie E; Makhlynets, Olga et al. (2014) Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis. J Biol Chem 289:6273-87
Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V et al. (2014) Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights. J Biol Chem 289:6259-72
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
Cotruvo Jr, Joseph A; Stich, Troy A; Britt, R David et al. (2013) Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate. J Am Chem Soc 135:4027-39
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
Boal, Amie K; Cotruvo Jr, Joseph A; Stubbe, Joanne et al. (2012) The dimanganese(II) site of Bacillus subtilis class Ib ribonucleotide reductase. Biochemistry 51:3861-71

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