Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase
The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with...
| Prif Awduron: | , , , , , |
|---|---|
| Awduron Eraill: | |
| Fformat: | Erthygl |
| Iaith: | en_US |
| Cyhoeddwyd: |
American Chemical Society (ACS)
2012
|
| Mynediad Ar-lein: | http://hdl.handle.net/1721.1/74085 https://orcid.org/0000-0003-1589-832X |
| _version_ | 1826216829071130624 |
|---|---|
| author | Zipse, Hendrik Artin, Erin Wnuk, Stanislaw Lohman, Gregory J. S. Martino, Debora Griffin, Robert Guy |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Zipse, Hendrik Artin, Erin Wnuk, Stanislaw Lohman, Gregory J. S. Martino, Debora Griffin, Robert Guy |
| author_sort | Zipse, Hendrik |
| collection | MIT |
| description | The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-[superscript 2]H], [2′-[superscript 2]H], [4′-[superscript 2]H], [5′-[superscript 2]H], [U−[superscript 13]C, [superscript 15]N], [U−[superscript 15]N], and [5,6 -[superscript 2]H] CDP and [β-[superscript 2]H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of [superscript 2]H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed. |
| first_indexed | 2024-09-23T16:54:04Z |
| format | Article |
| id | mit-1721.1/74085 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:54:04Z |
| publishDate | 2012 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/740852022-09-29T22:15:49Z Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase Zipse, Hendrik Artin, Erin Wnuk, Stanislaw Lohman, Gregory J. S. Martino, Debora Griffin, Robert Guy Massachusetts Institute of Technology. Department of Chemistry Francis Bitter Magnet Laboratory (Massachusetts Institute of Technology) Artin, Erin Lohman, Gregory J. S. Martino, Debora Griffin, Robert Guy The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-[superscript 2]H], [2′-[superscript 2]H], [4′-[superscript 2]H], [5′-[superscript 2]H], [U−[superscript 13]C, [superscript 15]N], [U−[superscript 15]N], and [5,6 -[superscript 2]H] CDP and [β-[superscript 2]H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of [superscript 2]H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed. National Institutes of Health (U.S.) (GM29595) (EB002804) (EB002026) 2012-10-18T17:11:49Z 2012-10-18T17:11:49Z 2008-12 2008-08 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/74085 Zipse, Hendrik et al. “Structure of the Nucleotide Radical Formed During Reaction of CDP/TTP with the E441Q-α2β2 of E. Coli Ribonucleotide Reductase.” Journal of the American Chemical Society 131.1 (2009): 200–211. Copyright © 2008 American Chemical Society https://orcid.org/0000-0003-1589-832X en_US http://dx.doi.org/10.1021/ja806693s Journal of the American Chemical Society Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) ACS |
| spellingShingle | Zipse, Hendrik Artin, Erin Wnuk, Stanislaw Lohman, Gregory J. S. Martino, Debora Griffin, Robert Guy Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title | Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title_full | Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title_fullStr | Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title_full_unstemmed | Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title_short | Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase |
| title_sort | structure of the nucleotide radical formed during reaction of cdp ttp with the e441q α2β2 of e coli ribonucleotide reductase |
| url | http://hdl.handle.net/1721.1/74085 https://orcid.org/0000-0003-1589-832X |
| work_keys_str_mv | AT zipsehendrik structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase AT artinerin structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase AT wnukstanislaw structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase AT lohmangregoryjs structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase AT martinodebora structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase AT griffinrobertguy structureofthenucleotideradicalformedduringreactionofcdpttpwiththee441qa2b2ofecoliribonucleotidereductase |