Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase

dc.contributor.authorJohnson, Sydney B.en
dc.contributor.authorLi, Haoen
dc.contributor.authorValentino, Hannahen
dc.contributor.authorSobrado, Pabloen
dc.date.accessioned2024-06-20T16:52:21Zen
dc.date.available2024-06-20T16:52:21Zen
dc.date.issued2024-05-23en
dc.description.abstractOxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a “cautious” flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme−substrate and enzyme− intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.en
dc.description.sponsorshipThis work was supported by the U.S. National Science Foundation under Grant CHE 2003658. Further financial support was provided by the College of Agriculture and Life Sciences and the Department of Biochemistry under the Graduate Teaching Scholars program at Virginia Tech.en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1021/acs.biochem.3c00656en
dc.identifier.urihttps://hdl.handle.net/10919/119476en
dc.identifier.volume63en
dc.language.isoenen
dc.publisherAmerican Chemical Societyen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleMechanism of Nitrone Formation by a Flavin-Dependent Monooxygenaseen
dc.title.serialBiochemistryen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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