ArticleProceedings of the National Academy of Sciences of the United States of America2013
EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 21 citations in OpenAlex.
- EPR Characterization of the BlsE Substrate Radical Offers Insight into the Determinants of Reaction Outcome that Distinguish Radical SAM Dioldehydratases from Dehydrogenases.Journal of the American Chemical Society · 2025Article
- Mechanistic Investigation of 1,2-Diol Dehydration of Paromamine Catalyzed by the RadicalJournal of the American Chemical Society · 2021Article
- Measurement of Net Rate Constants from Enzyme Progress Curves without Curve Fitting.Biochemistry · 2019Article
- Mechanistic Implications of the Deamination of TDP-4-amino-4-deoxy-d-fucose Catalyzed by the Radical SAM Enzyme DesII.Biochemistry · 2018Article
- Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl-l-methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration.Journal of the American Chemical Society · 2017Article
- Theory and Application of the Relationship Between Steady-State Isotope Effects on Enzyme Intermediate Concentrations and Net Rate Constants.Methods in enzymology · 2017Article
- C3'-Deoxygenation of Paromamine Catalyzed by a Radical S-Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3.Angewandte Chemie (International ed. in English) · 2016Article
- Mechanistic Investigation of the Radical S-Adenosyl-L-methionine Enzyme DesII Using Fluorinated Analogues.Journal of the American Chemical Society · 2015Article
- Mechanistic Enzymology of the Radical SAM Enzyme DesII.Israel journal of chemistry · 2015Article
- Spore photoproduct lyase: the known, the controversial, and the unknown.The Journal of biological chemistry · 2015Review
- Mechanistic studies of the radical S-adenosylmethionine enzyme DesII with TDP-D-fucose.Angewandte Chemie (International ed. in English) · 2015Article
- Radical S-adenosylmethionine enzymes.Chemical reviews · 2014Review
- X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry.Proceedings of the National Academy of Sciences of the United States of America · 2013Article
- The enzyme-mediated direct reversal of a dithymine photoproduct in germinating endospores.International journal of molecular sciences · 2013Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0.22 ± 0.03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1.8 ± 0.2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.
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