Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch
Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tauto...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2015
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| Online Access: | http://hdl.handle.net/1721.1/99350 https://orcid.org/0000-0002-8241-4834 https://orcid.org/0000-0002-2196-5691 |
| _version_ | 1826188490200580096 |
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| author | Singh, Vipender Peng, Chunte Sam Li, Deyu Mitra, Koyel Silvestre, Katherine J. Tokmakoff, Andrei Essigmann, John M. |
| author2 | Massachusetts Institute of Technology. Center for Environmental Health Sciences |
| author_facet | Massachusetts Institute of Technology. Center for Environmental Health Sciences Singh, Vipender Peng, Chunte Sam Li, Deyu Mitra, Koyel Silvestre, Katherine J. Tokmakoff, Andrei Essigmann, John M. |
| author_sort | Singh, Vipender |
| collection | MIT |
| description | Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4′-keto tautomer that was likely protonated at the N1′-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected. |
| first_indexed | 2024-09-23T08:00:22Z |
| format | Article |
| id | mit-1721.1/99350 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:00:22Z |
| publishDate | 2015 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/993502022-09-23T10:12:53Z Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch Singh, Vipender Peng, Chunte Sam Li, Deyu Mitra, Koyel Silvestre, Katherine J. Tokmakoff, Andrei Essigmann, John M. Massachusetts Institute of Technology. Center for Environmental Health Sciences Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Chemistry Singh, Vipender Peng, Chunte Sam Li, Deyu Mitra, Koyel Silvestre, Katherine J. Tokmakoff, Andrei Essigmann, John M. Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4′-keto tautomer that was likely protonated at the N1′-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected. National Institutes of Health (U.S.) (Grant CA080024) National Institutes of Health (U.S.) (Grant CA26731) National Institutes of Health (U.S.) (Grant ES002109) National Institutes of Health (U.S.) (Grant ES007020) National Science Foundation (U.S.) (Grant CHE-1212557) Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institutes of Health (U.S.) Center Grant P30-ES002109) Massachusetts Institute of Technology. Laser Biomedical Research Center (National Institutes of Health (U.S.) Center Grant P41-EB015871) National Institutes of Health (U.S.) (Traineeship T32 ES007020) Massachusetts Institute of Technology (Poitras Pre-Doctoral Fellowship) 2015-10-16T12:42:12Z 2015-10-16T12:42:12Z 2013-10 2013-08 Article http://purl.org/eprint/type/JournalArticle 1554-8929 1554-8937 http://hdl.handle.net/1721.1/99350 Singh, Vipender, Chunte Sam Peng, Deyu Li, Koyel Mitra, Katherine J. Silvestre, Andrei Tokmakoff, and John M. Essigmann. “Direct Observation of Multiple Tautomers of Oxythiamine and Their Recognition by the Thiamine Pyrophosphate Riboswitch.” ACS Chemical Biology 9, no. 1 (January 17, 2014): 227–36. https://orcid.org/0000-0002-8241-4834 https://orcid.org/0000-0002-2196-5691 en_US http://dx.doi.org/10.1021/cb400581f ACS Chemical Biology 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) PMC |
| spellingShingle | Singh, Vipender Peng, Chunte Sam Li, Deyu Mitra, Koyel Silvestre, Katherine J. Tokmakoff, Andrei Essigmann, John M. Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title | Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title_full | Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title_fullStr | Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title_full_unstemmed | Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title_short | Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch |
| title_sort | direct observation of multiple tautomers of oxythiamine and their recognition by the thiamine pyrophosphate riboswitch |
| url | http://hdl.handle.net/1721.1/99350 https://orcid.org/0000-0002-8241-4834 https://orcid.org/0000-0002-2196-5691 |
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