Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

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dc.contributor.authorBuren, Stefanen
dc.contributor.authorPratt, Katelinen
dc.contributor.authorJiang, Xien
dc.contributor.authorGuo, Yisongen
dc.contributor.authorJimenez-Vicente, Emilioen
dc.contributor.authorEchavarri-Erasun, Carlosen
dc.contributor.authorDean, Dennis R.en
dc.contributor.authorSaaem, Ishtiaqen
dc.contributor.authorGordon, D. Benjaminen
dc.contributor.authorVoigt, Christopher A.en
dc.contributor.authorRubio, Luis M.en
dc.date.accessioned2021-10-07T13:50:52Zen
dc.date.available2021-10-07T13:50:52Zen
dc.date.issued2019-12-10en
dc.date.updated2021-10-07T13:50:49Zen
dc.description.abstractThe radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.en
dc.description.versionPublished versionen
dc.format.extentPages 25078-25086en
dc.format.extent9 page(s)en
dc.identifier.doihttps://doi.org/10.1073/pnas.1904903116en
dc.identifier.eissn1091-6490en
dc.identifier.issn0027-8424en
dc.identifier.issue50en
dc.identifier.orcidDean, Dennis [0000-0001-8960-6196]en
dc.identifier.other1904903116 (PII)en
dc.identifier.pmid31767756en
dc.identifier.urihttp://hdl.handle.net/10919/105194en
dc.identifier.volume116en
dc.language.isoenen
dc.publisherNational Academy of Sciencesen
dc.relation.urihttp://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000502577500027&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=930d57c9ac61a043676db62af60056c1en
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectnitrogen fixationen
dc.subjectsynthetic biologyen
dc.subjectnif genesen
dc.subjectcombinatorial designen
dc.subjectmitochondriaen
dc.subjectIRON-MOLYBDENUM COFACTORen
dc.subjectIN-VITRO SYNTHESISen
dc.subjectAZOTOBACTER-VINELANDIIen
dc.subjectMOFE-PROTEINen
dc.subjectPURIFICATIONen
dc.subjectEXPRESSIONen
dc.subjectCLUSTERen
dc.subjectSULFURen
dc.subjectIDENTIFICATIONen
dc.subjectPRODUCTen
dc.subjectcombinatorial designen
dc.subjectmitochondriaen
dc.subjectnif genesen
dc.subjectnitrogen fixationen
dc.subjectsynthetic biologyen
dc.subject.meshMitochondriaen
dc.subject.meshAzotobacter vinelandiien
dc.subject.meshSaccharomyces cerevisiaeen
dc.subject.meshIron Compoundsen
dc.subject.meshNitrogenaseen
dc.subject.meshBacterial Proteinsen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshNitrogen Fixationen
dc.subject.meshMetabolic Networks and Pathwaysen
dc.subject.meshSynthetic Biologyen
dc.subject.meshMethanocaldococcusen
dc.titleBiosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiaeen
dc.title.serialProceedings of the National Academy of Sciences of the United States of Americaen
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 Distinguished Professorsen
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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