A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates
<jats:p>Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucl...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022
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| Online Access: | https://hdl.handle.net/1721.1/144117 |
| _version_ | 1826207708630482944 |
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| author | Levitz, Talya S Andree, Gisele A Jonnalagadda, Rohan Dawson, Christopher D Bjork, Rebekah E Drennan, Catherine L |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Levitz, Talya S Andree, Gisele A Jonnalagadda, Rohan Dawson, Christopher D Bjork, Rebekah E Drennan, Catherine L |
| author_sort | Levitz, Talya S |
| collection | MIT |
| description | <jats:p>Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical <jats:italic>Escherichia coli</jats:italic> aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. <jats:italic>Streptococcus thermophilus</jats:italic> RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the <jats:italic>E</jats:italic>. <jats:italic>coli</jats:italic> class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.</jats:p> |
| first_indexed | 2024-09-23T13:54:04Z |
| format | Article |
| id | mit-1721.1/144117 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T13:54:04Z |
| publishDate | 2022 |
| publisher | Public Library of Science (PLoS) |
| record_format | dspace |
| spelling | mit-1721.1/1441172023-07-19T20:30:37Z A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates Levitz, Talya S Andree, Gisele A Jonnalagadda, Rohan Dawson, Christopher D Bjork, Rebekah E Drennan, Catherine L Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Howard Hughes Medical Institute Massachusetts Institute of Technology. Center for Environmental Health Sciences <jats:p>Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical <jats:italic>Escherichia coli</jats:italic> aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. <jats:italic>Streptococcus thermophilus</jats:italic> RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the <jats:italic>E</jats:italic>. <jats:italic>coli</jats:italic> class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.</jats:p> 2022-07-29T12:47:57Z 2022-07-29T12:47:57Z 2022 2022-07-29T12:08:17Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/144117 Levitz, Talya S, Andree, Gisele A, Jonnalagadda, Rohan, Dawson, Christopher D, Bjork, Rebekah E et al. 2022. "A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates." PLOS ONE, 17 (6). en 10.1371/journal.pone.0269572 PLOS ONE Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Public Library of Science (PLoS) PLoS |
| spellingShingle | Levitz, Talya S Andree, Gisele A Jonnalagadda, Rohan Dawson, Christopher D Bjork, Rebekah E Drennan, Catherine L A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title | A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title_full | A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title_fullStr | A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title_full_unstemmed | A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title_short | A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| title_sort | rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates |
| url | https://hdl.handle.net/1721.1/144117 |
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