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Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

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dc.contributor.authorCampbell, Ashley C.en
dc.contributor.authorStiers, Kyle M.en
dc.contributor.authorDel Campo, Julia S. Martinen
dc.contributor.authorMehra-Chaudhary, Ritchaen
dc.contributor.authorSobrado, Pabloen
dc.contributor.authorTanner, John J.en
dc.contributor.departmentBiochemistryen
dc.date.accessioned2021-02-16T13:49:46Zen
dc.date.available2021-02-16T13:49:46Zen
dc.date.issued2020-09-18en
dc.description.abstractThe siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase fromAspergillus fumigatusis a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is anN-monooxygenase that catalyzes the NADPH-dependent hydroxylation ofl-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) orl-ornithine bound (resting state). The resting state structure reveals a newoutactive site conformation characterized by large rotations of the FAD isoalloxazine around the C1-' C2 ' and N10-C1 ' bonds, coupled to a 10-angstrom movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to theinconformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) andl-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of theoutconformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.en
dc.description.notesResearch reported in this publication was supported by the National Science Foundation Grants CHE-2003658 (to P. S) and CHE-2003986 (to J. J. T). A. C. C. was funded in part by a Fulbright Science & Innovation Graduate Award.en
dc.description.sponsorshipNational Science FoundationNational Science Foundation (NSF) [CHE-2003658, CHE-2003986]; Fulbright Science & Innovation Graduate Awarden
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1074/jbc.RA120.014750en
dc.identifier.eissn1083-351Xen
dc.identifier.issn0021-9258en
dc.identifier.issue38en
dc.identifier.pmid32723870en
dc.identifier.urihttp://hdl.handle.net/10919/102380en
dc.identifier.volume295en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectX-ray crystallographyen
dc.subjectenzyme structureen
dc.subjectflavoproteinen
dc.subjectenzyme kineticsen
dc.subjectsite-directed mutagenesisen
dc.subjectsiderophoreen
dc.subjectflavin-dependent monooxygenasesen
dc.subjectornithine hydroxylaseen
dc.titleTrapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamicsen
dc.title.serialJournal of Biological Chemistryen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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