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Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation
[Image: see text] Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide pro...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139161/ https://www.ncbi.nlm.nih.gov/pubmed/25068636 http://dx.doi.org/10.1021/bi500648n |
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author | Conrad, John A. Ortiz-Maldonado, Mariliz Hoppe, Samuel W. Palfey, Bruce A. |
author_facet | Conrad, John A. Ortiz-Maldonado, Mariliz Hoppe, Samuel W. Palfey, Bruce A. |
author_sort | Conrad, John A. |
collection | PubMed |
description | [Image: see text] Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH(2)THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis. |
format | Online Article Text |
id | pubmed-4139161 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-41391612015-07-28 Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation Conrad, John A. Ortiz-Maldonado, Mariliz Hoppe, Samuel W. Palfey, Bruce A. Biochemistry [Image: see text] Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH(2)THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis. American Chemical Society 2014-07-28 2014-08-19 /pmc/articles/PMC4139161/ /pubmed/25068636 http://dx.doi.org/10.1021/bi500648n Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Conrad, John A. Ortiz-Maldonado, Mariliz Hoppe, Samuel W. Palfey, Bruce A. Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title | Detection of Intermediates in the Oxidative Half-Reaction
of the FAD-Dependent Thymidylate Synthase from Thermotoga
maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title_full | Detection of Intermediates in the Oxidative Half-Reaction
of the FAD-Dependent Thymidylate Synthase from Thermotoga
maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title_fullStr | Detection of Intermediates in the Oxidative Half-Reaction
of the FAD-Dependent Thymidylate Synthase from Thermotoga
maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title_full_unstemmed | Detection of Intermediates in the Oxidative Half-Reaction
of the FAD-Dependent Thymidylate Synthase from Thermotoga
maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title_short | Detection of Intermediates in the Oxidative Half-Reaction
of the FAD-Dependent Thymidylate Synthase from Thermotoga
maritima: Carbon Transfer without Covalent Pyrimidine Activation |
title_sort | detection of intermediates in the oxidative half-reaction
of the fad-dependent thymidylate synthase from thermotoga
maritima: carbon transfer without covalent pyrimidine activation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139161/ https://www.ncbi.nlm.nih.gov/pubmed/25068636 http://dx.doi.org/10.1021/bi500648n |
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