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Bacillus subtilis Class Ib Ribonucleotide Reductase Is a Dimanganese(III)-Tyrosyl Radical Enzyme

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dc.contributor.author Zhang, Yan
dc.contributor.author Stubbe, JoAnne
dc.date.accessioned 2012-08-14T21:30:07Z
dc.date.available 2012-08-14T21:30:07Z
dc.date.issued 2011-05
dc.date.submitted 2011-03
dc.identifier.issn 0006-2960
dc.identifier.issn 1520-4995
dc.identifier.uri http://hdl.handle.net/1721.1/72129
dc.description.abstract Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)[subscript 2-]Y[superscript•] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O[subscript 2]. Apo-rNrdF loaded using 4 Mn(II)/β[subscript 2], O[subscript 2], and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y[superscript•] [Mn(III)[subscript 2-]Y[superscript•]] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)[subscript 2-]Y[superscript•] and Fe(III)[subscript 2-]Y[superscript•] rNrdF generate dCDP at rates of 132 and 10 nmol min[superscript –1] mg[superscript –1], respectively (both normalized for 1 Y[superscript•]/β[subscript 2]). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)[subscript 2-]Y[superscript•] center with 2 Mn/β[subscript 2] and 0.5 Y[superscript•]/β[subscript 2] and an activity of 318–363 nmol min[superscript –1] mg[superscript –1] (normalized for 1 Y[superscript•]/β[subscript 2]). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)[subscript 2]-Y[superscript•] enzyme. en_US
dc.description.sponsorship National Institutes of Health (U.S.) (Grant number GM81393) en_US
dc.language.iso en_US
dc.publisher American Chemical Society (ACS) en_US
dc.relation.isversionof http://dx.doi.org/10.1021/bi200348q en_US
dc.rights Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. en_US
dc.source PMC en_US
dc.title Bacillus subtilis Class Ib Ribonucleotide Reductase Is a Dimanganese(III)-Tyrosyl Radical Enzyme en_US
dc.type Article en_US
dc.identifier.citation Zhang, Yan, and JoAnne Stubbe. “Bacillus Subtilis Class Ib Ribonucleotide Reductase Is a Dimanganese(III)-Tyrosyl Radical Enzyme.” Biochemistry 50.25 (2011): 5615–5623. en_US
dc.contributor.department Massachusetts Institute of Technology. Department of Chemistry en_US
dc.contributor.department Massachusetts Institute of Technology. Department of Biology en_US
dc.contributor.approver Stubbe, JoAnne
dc.contributor.mitauthor Zhang, Yan
dc.contributor.mitauthor Stubbe, JoAnne
dc.relation.journal Biochemistry en_US
dc.identifier.mitlicense PUBLISHER_POLICY en_US
dc.eprint.version Author's final manuscript en_US
dc.type.uri http://purl.org/eprint/type/JournalArticle en_US
eprint.status http://purl.org/eprint/status/PeerReviewed en_US
dspace.orderedauthors Zhang, Yan; Stubbe, JoAnne en


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