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Structural Determinants of Flavin Dynamics in a Class B Monooxygenase

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dc.contributor.authorCampbell, Ashley C.en
dc.contributor.authorRobinson, Reederen
dc.contributor.authorMena-Aguilar, Didieren
dc.contributor.authorSobrado, Pabloen
dc.contributor.authorTanner, John J.en
dc.date.accessioned2022-01-13T02:24:45Zen
dc.date.available2022-01-13T02:24:45Zen
dc.date.issued2020-12-08en
dc.date.updated2022-01-13T02:24:37Zen
dc.description.abstractThe ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-l-ornithine and NADP+. Crystal structures were determined for M101A in the resting state and complexed with NADP+. The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP+ complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP+ in preparation for a new catalytic cycle.en
dc.description.versionAccepted versionen
dc.format.extentPages 4609-4616en
dc.format.extent8 page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1021/acs.biochem.0c00783en
dc.identifier.eissn1520-4995en
dc.identifier.issn0006-2960en
dc.identifier.issue48en
dc.identifier.orcidSobrado, Pablo [0000-0003-1494-5382]en
dc.identifier.pmid33226785en
dc.identifier.urihttp://hdl.handle.net/10919/107583en
dc.identifier.volume59en
dc.language.isoenen
dc.publisherAmerican Chemical Societyen
dc.relation.urihttp://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000599322700010&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=930d57c9ac61a043676db62af60056c1en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectLife Sciences & Biomedicineen
dc.subjectBiochemistry & Molecular Biologyen
dc.subjectORNITHINE-HYDROXYLASEen
dc.subjectSIDEROPHOREen
dc.subjectMECHANISMen
dc.subjectSIDAen
dc.subjectVALIDATIONen
dc.subjectCATALYSISen
dc.subjectNADP(H)en
dc.subject0304 Medicinal and Biomolecular Chemistryen
dc.subject0601 Biochemistry and Cell Biologyen
dc.subject1101 Medical Biochemistry and Metabolomicsen
dc.subjectBiochemistry & Molecular Biologyen
dc.subject.meshAspergillus fumigatusen
dc.subject.meshFlavin-Adenine Dinucleotideen
dc.subject.meshMixed Function Oxygenasesen
dc.subject.meshFlavinsen
dc.subject.meshFungal Proteinsen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshSiderophoresen
dc.subject.meshCrystallography, X-Rayen
dc.subject.meshMutagenesis, Site-Directeden
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshProtein Conformationen
dc.subject.meshOxidation-Reductionen
dc.subject.meshKineticsen
dc.subject.meshModels, Molecularen
dc.subject.meshStatic Electricityen
dc.titleStructural Determinants of Flavin Dynamics in a Class B Monooxygenaseen
dc.title.serialBiochemistryen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherArticleen
dc.type.otherJournalen
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Biochemistryen
pubs.organisational-group/Virginia Tech/University Research Institutesen
pubs.organisational-group/Virginia Tech/University Research Institutes/Fralin Life Sciencesen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen
pubs.organisational-group/Virginia Tech/University Research Institutes/Fralin Life Sciences/Durelle Scotten

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