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

Author(s)
Zhang, Yan; Stubbe, JoAnne
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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.
Date issued
2011-05
URI
http://hdl.handle.net/1721.1/72129
Department
Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
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.
Version: Author's final manuscript
ISSN
0006-2960
1520-4995

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