title: Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.
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)(2)-Y(•)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/β(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(•) [Mn(III)(2)-Y(•)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(•) and Fe(III)(2)-Y(•) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(•)/β(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)(2)-Y(•) center with 2 Mn/β(2) and 0.5 Y(•)/β(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(•)/β(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(•) enzyme.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Joanne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005609:Free Radicals / Q000737:chemistry; D017895:Manganese Compounds / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics; D009876:Operon / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005609:Free Radicals; D017895:Manganese Compounds; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D012264:Ribonucleotide Reductases; D014443:Tyrosine; C534806:YmdB protein, E coli; C093564:Neurogenic differentiation factor 1; C532351:NrdE protein, E coli; C114872:NrdF protein, bacteria