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Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis

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dc.contributor.authorValentino, Hannahen
dc.contributor.authorSobrado, Pabloen
dc.date.accessioned2022-01-13T02:18:54Zen
dc.date.available2022-01-13T02:18:54Zen
dc.date.issued2021-09-28en
dc.date.updated2022-01-13T02:18:37Zen
dc.description.abstractN-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in someStreptomycessp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higherkcat/KMvalue for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where thekredis rate-limiting, FzmM exhibits a very fastkred(50 ± 0.01 s-1at 4 °C) with NADPH. NADPH binds at aKDvalue of ∼400 μM, and hydride transfer occurs withpro-Rstereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than thekcat. This reaction is enhanced in the presence of Asp with a slightly fasterkoxthan thekcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.en
dc.description.versionAccepted versionen
dc.format.extentPages 2851-2864en
dc.format.extent14 page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1021/acs.biochem.1c00512en
dc.identifier.eissn1520-4995en
dc.identifier.issn0006-2960en
dc.identifier.issue38en
dc.identifier.orcidSobrado, Pablo [0000-0003-1494-5382]en
dc.identifier.pmid34516102en
dc.identifier.urihttp://hdl.handle.net/10919/107580en
dc.identifier.volume60en
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:000703528500002&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.subjectSTRUCTURAL DETERMINANTSen
dc.subjectPSEUDOMONAS-AERUGINOSAen
dc.subjectKINETIC MECHANISMen
dc.subjectOXYGEN ACTIVATIONen
dc.subjectFLAVINen
dc.subjectMONOOXYGENASEen
dc.subjectSUBSTRATEen
dc.subjectIDENTIFICATIONen
dc.subjectSELECTIVITYen
dc.subject0304 Medicinal and Biomolecular Chemistryen
dc.subject0601 Biochemistry and Cell Biologyen
dc.subject1101 Medical Biochemistry and Metabolomicsen
dc.subject.meshSuccinic Aciden
dc.subject.meshHydrazinesen
dc.subject.meshDinitrocresolsen
dc.subject.meshOrganophosphorus Compoundsen
dc.subject.meshFlavin-Adenine Dinucleotideen
dc.subject.meshNADPen
dc.subject.meshMixed Function Oxygenasesen
dc.subject.meshFlavinsen
dc.subject.meshOxidation-Reductionen
dc.subject.meshHydroxylationen
dc.subject.meshKineticsen
dc.titleCharacterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesisen
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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