Ribonucleotide reductases (RNRs) provide deoxyribonucleotides for DNA synthesis and repair. The enzymes employ a conserved free-radical mechanism. Class I RNRs, including the human and Herpes Simplex Virus I enzymes, use a stable tyrosyl radical to initiate this mechanism and are validated drug targets. Several of the drugs function (at least in part) by reducing the tyrosyl radical. The tyrosyl radical is introduced into the enzyme by reaction of a di-iron(II) center with O2. Class I RNRs found in important human pathogens such as Chlamydia trachomatis and Mycobacterium tuberculosis lack the tyrosyl radical. The C. trachomatis RNR is, nevertheless, active. We recently showed that the C. trachomatis RNR uses a stable Mn(IV)/Fe(III) cofactor in place of the tyrosyl radical to initiates its reaction. The cofactor undergoes reduction to the Mn(III)/Fe(III) form to generate a protein radical that abstracts a hydrogen atom from the substrate. The Ct RNR is the first example of a manganese-dependent RNR, and its cofactor is the first example of a Mn/Fe redox center in biology. The cofactor is introduced, analogously to the tyrosyl radical in the conventional class I RNRs, by reaction of the reduced [Mn(II)/Fe(II)] metal center with O2. In this reaction, a Mn(IV)/Fe(IV) accumulates to a high level. In this project, we will elucidate the mechanisms of the formation and catalytic function of this novel cofactor. We will define the structures of its Mn(II)/Fe(II), Mn(IIII)/Fe(III), Mn(IV)/Fe(III) and Mn(IV)/Fe(IV) states by spectroscopic and computational methods and x-ray crystallography. We will understand how the protein protects the oxidized cofactor from adventitious reduction but then allows it to be reduced at the appropriate time to form the hydrogen-abstracting protein radical. We will study its chemical reactivity to uncover unique vulnerabilities that might be exploited in design of new drugs against the pathogens that use this type of RNR. Finally, we will compare the structures of closely related pairs of RNRs, of which one uses the standard tyrosyl radical and the other the novel Mn(IV)/Fe(III) cofactor, for clues to the design of both systems and the evolution of one from another. We will then attempt to use these clues to rationally convert one type of RNR into the other by changing crucial amino acids.

Public Health Relevance

The enzyme ribonucleotide reductase (RNR) catalyzes the key step in DNA biosynthesis of all organisms and is a validated target for treatment of cancer and viral diseases. We recently reported that the class Ic RNR from the human pathogen Chlamydia trachomatis uses a novel redox cofactor (a heterobinuclear Mn/Fe cluster) to initiate catalysis. The structure and mechanism of this novel RNR will be elucidated to facilitate the rational development of class Ic RNR inhibitors that could be used to treat diseases caused by C. trachomatis and several other human pathogens (e.g. Chlamydia pneumoniae and Mycobacterium tuberculosis).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Anderson, Vernon
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Martinie, Ryan J; Blaesi, Elizabeth J; Krebs, Carsten et al. (2017) Evidence for a Di-?-oxo Diamond Core in the Mn(IV)/Fe(IV) Activation Intermediate of Ribonucleotide Reductase from Chlamydia trachomatis. J Am Chem Soc 139:1950-1957
Livada, Jovan; Martinie, Ryan J; Dassama, Laura M K et al. (2015) Direct Measurement of the Radical Translocation Distance in the Class I Ribonucleotide Reductase from Chlamydia trachomatis. J Phys Chem B 119:13777-84
Krebs, Carsten; Dassama, Laura M K; Matthews, Megan L et al. (2013) Novel Approaches for the Accumulation of Oxygenated Intermediates to Multi-Millimolar Concentrations. Coord Chem Rev 257:
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
Kwak, Yeonju; Jiang, Wei; Dassama, Laura M K et al. (2013) Geometric and electronic structure of the Mn(IV)Fe(III) cofactor in class Ic ribonucleotide reductase: correlation to the class Ia binuclear non-heme iron enzyme. J Am Chem Soc 135:17573-84
Dassama, Laura M K; Krebs, Carsten; Bollinger Jr, J Martin et al. (2013) Structural basis for assembly of the Mn(IV)/Fe(III) cofactor in the class Ic ribonucleotide reductase from Chlamydia trachomatis. Biochemistry 52:6424-36
Dassama, Laura M K; Silakov, Alexey; Krest, Courtney M et al. (2013) A 2.8 A Fe-Fe separation in the Fe2(III/IV) intermediate, X, from Escherichia coli ribonucleotide reductase. J Am Chem Soc 135:16758-61
Pandelia, Maria E; Li, Ning; Nørgaard, Hanne et al. (2013) Substrate-triggered addition of dioxygen to the diferrous cofactor of aldehyde-deformylating oxygenase to form a diferric-peroxide intermediate. J Am Chem Soc 135:15801-12
Dassama, Laura M K; Yosca, Timothy H; Conner, Denise A et al. (2012) O(2)-evolving chlorite dismutase as a tool for studying O(2)-utilizing enzymes. Biochemistry 51:1607-16
Dassama, Laura M K; Boal, Amie K; Krebs, Carsten et al. (2012) Evidence that the ? subunit of Chlamydia trachomatis ribonucleotide reductase is active with the manganese ion of its manganese(IV)/iron(III) cofactor in site 1. J Am Chem Soc 134:2520-3

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