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Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation

2010-01-01Artigo científico Acesso aberto
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dc.contributor.authorMarohnic, Christopher C
dc.contributor.authorPanda, Satya P.
dc.contributor.authorMcCammon, Karen M.
dc.contributor.authorRueff, J.
dc.contributor.authorRueff, Jose
dc.contributor.authorMasters, Bettie Sue Sile
dc.contributor.authorKranendonk, Michel
dc.contributor.institutionNOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM)
dc.contributor.institutionCentro de Investigação em Genética Molecular Humana (CIGMH)
dc.contributor.pblASPET - American Society for Pharmacology and Experimental Therapeutics
dc.date.accessioned2022-07-19T22:19:01Z
dc.date.available2022-07-21T00:31:28Z
dc.date.embargoedUntil2022-07-20
dc.date.issued2010-01-01
dc.descriptionThis work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM081568]; the Robert A. Welch Foundation [Endowed Chair AQ-0012] (to B. S. S. M.); and the Fundacao para a Ciencia e a Tecnologia (Portugal) [Grant PTDC/SAU-GMG/71911/2006].
dc.description.abstractPatients with congenital adrenal hyperplasia, exhibiting combined CYP17 and CYP21 deficiency, were shown by Arlt et al. (2004) to harbor a 541T -> G mutation in exon 5 of POR (encoding NADPH-cytochrome P450 reductase, CYPOR), which resulted in a Y181D substitution that obliterated electron transfer capacity. Using bacterial expression models, we examined catalytic and physical properties of the human CYPOR Y181D variant. As purified, Y181D lacked flavin mononucleotide (FMN) and NADPH-cytochrome c reductase (NCR) activity but retained normal flavin adenine dinucleotide binding and NADPH utilization. Titration of the purified protein with FMN restored 64% of wild-type (WT) NCR activity in Y181D with an activation constant of similar to 2 mu M. As determined by FMN fluorescence quenching, Y181D had K-d(FMN) = 7.3 mu M. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of similar to 1:10 in the engineered MK_1A2_POR Escherichia coli strain, showed the compromised capacity of Y181D to support CYP1A2-catalyzed metabolism of the procarcinogens 2-aminoanthracene, 2-amino-3-methylimidazo(4,5-f)quinoline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Isolated MK1A2_POR membranes confirmed FMN stimulation of Y181D NCR activity with a 1.6 mu M activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_PORY181D membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_PORWT membranes with a 1.2 mu M FMN activation constant. Therefore, we conclude that compromised FMN binding is the specific molecular defect causing POR deficiency in patients with Y181D mutation and that this defect, in large part, can be overcome in vitro by FMN addition.en
dc.description.versionpublishersversion
dc.description.versionpublished
dc.format.extent1205600
dc.identifier.doi10.1124/dmd.109.030445
dc.identifier.issn0090-9556
dc.identifier.otherPURE: 503161
dc.identifier.otherPURE UUID: 7c74c86f-c274-4391-97a9-c236f0dee985
dc.identifier.otherresearchoutputwizard: 14782
dc.identifier.otherPubMed: 19884324
dc.identifier.otherWOS: 000273726900017
dc.identifier.otherScopus: 76149123185
dc.identifier.urihttp://hdl.handle.net/10362/142135
dc.language.isoeng
dc.peerreviewedyes
dc.relationinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/71911/PT
dc.relationThe study of Antley-Bixler Syndrome related mutations of cytochrome P450 oxidoreductase: CYPOR polymorphism and the human cytochrome P450 enzyme complex
dc.subjectstrain
dc.subjectantley-bixler-syndrome
dc.subjectescherichia-coli
dc.subjectsteroidogenesis
dc.subjecttester
dc.subjectdisordered
dc.subjectp450
dc.subjectp-450
dc.subjectreductase
dc.subjectfmn-binding
dc.subjectsystem
dc.subjectrat-liver
dc.subjectdomain
dc.subjectmutant
dc.subjectoxidoreductase
dc.subjectriboflavin
dc.subjectANTLEY-BIXLER-SYNDROME
dc.subjectMUTANT P450 OXIDOREDUCTASE
dc.subjectFMN-BINDING DOMAIN
dc.subjectESCHERICHIA-COLI
dc.subjectDISORDERED STEROIDOGENESIS
dc.subjectP-450 REDUCTASE
dc.subjectTESTER STRAIN
dc.subjectRAT-LIVER
dc.subjectSYSTEM
dc.subjectRIBOFLAVIN
dc.titleHuman Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutationen
dc.title.subtitleMolecular Consequences and Rescue of Defecten
dc.typejournal article
degois.publication.firstPage332
degois.publication.issue2
degois.publication.lastPage340
degois.publication.titleDrug Metabolism And Disposition
degois.publication.volume38
dspace.entity.typePublication
oaire.awardNumberPTDC/SAU-GMG/71911/2006
oaire.awardTitleThe study of Antley-Bixler Syndrome related mutations of cytochrome P450 oxidoreductase: CYPOR polymorphism and the human cytochrome P450 enzyme complex
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FSAU-GMG%2F71911%2F2006/PT
oaire.fundingStream3599-PPCDT
person.familyNameRueff
person.givenNameJose
person.identifier793666
person.identifier.ciencia-id0E15-908D-EA21
person.identifier.orcid0000-0002-8456-7295
person.identifier.ridE-6426-2013
person.identifier.scopus-author-id7006536439
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccess
relation.isAuthorOfPublication91a3b5ac-0328-498d-8cb8-08555b202306
relation.isAuthorOfPublication.latestForDiscovery91a3b5ac-0328-498d-8cb8-08555b202306
relation.isProjectOfPublicationd5dc0bfd-7a4e-4495-b184-9cc106c168e1
relation.isProjectOfPublication.latestForDiscoveryd5dc0bfd-7a4e-4495-b184-9cc106c168e1

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