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Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii

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dc.contributor.authorLyons, Noah S.en
dc.contributor.authorBogner, Alexandra N.en
dc.contributor.authorTanner, John J.en
dc.contributor.authorSobrado, Pabloen
dc.date.accessioned2023-01-23T15:51:54Zen
dc.date.available2023-01-23T15:51:54Zen
dc.date.issued2022-11-15en
dc.date.updated2023-01-22T18:32:43Zen
dc.description.abstractAcinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+en
dc.description.versionAccepted versionen
dc.format.extentPages 2607-2620en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1021/acs.biochem.2c00493en
dc.identifier.eissn1520-4995en
dc.identifier.issn0006-2960en
dc.identifier.issue22en
dc.identifier.orcidSobrado, Pablo [0000-0003-1494-5382]en
dc.identifier.pmid36314559en
dc.identifier.urihttp://hdl.handle.net/10919/113361en
dc.identifier.volume61en
dc.language.isoenen
dc.publisherAmerican Chemical Societyen
dc.relation.urihttps://www.ncbi.nlm.nih.gov/pubmed/36314559en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectInfectious Diseasesen
dc.subject.meshAcinetobacter baumanniien
dc.subject.meshCadaverineen
dc.subject.meshPutrescineen
dc.subject.meshNADPen
dc.subject.meshMixed Function Oxygenasesen
dc.subject.meshFlavinsen
dc.subject.meshOrnithineen
dc.subject.meshSiderophoresen
dc.subject.meshAnti-Bacterial Agentsen
dc.subject.meshKineticsen
dc.titleKinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from <i>Acinetobacter baumannii</i>en
dc.title.serialBiochemistryen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherJournal Articleen
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/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

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