Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.
Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.
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DOI：
10.1021/bi200348q
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发表时间：
2011-06-28
期刊：
影响因子：
2.9
通讯作者：
Stubbe J
中科院分区：
文献类型：
--
作者：
Zhang Y;Stubbe J
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 E. coli. As isolated, recombinant (r) rNrdF contained a diferric-tyrosyl radical (Fe(III)2-Y•) cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O2. Apo-rNrdF loaded using 4 Mn(II)/β2, O2 and reduced NrdI (a flavodoxin), can form a dimanganese(III)-Y• (Mn(III)2-Y•) cofactor. In the presence of rNrdE/ATP/CDP, Mn(III)2-Y• and Fe(III)2-Y• rNrdF generate dCDP at 132 and 10 nmol min-1 mg-1 respectively (both normalized for 1 Y•/β2). 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 LB medium, with levels of NrdE and NrdF elevated 35 fold relative to the wild type (wt) strain. NrdE and NrdF co-purified 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)2-Y• center with 2 Mn and 0.5 Y•/β2 and activity of 318-363 nmol min-1 mg-1 (normalized for 1 Y•/β2). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)2-Y• enzyme.
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