title: Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.
abstract: The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn(III)2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn(III)2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min(-1) mg(-1)). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an "active" RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn(III)2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β2. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min(-1) mg(-1) using a 1:1 mixture of NrdE:Mn(III)2-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 μM), NrdE is a monomer (α) and Mn(III)2-Y· NrdF is a dimer (β2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.
authors.affiliation: Departments of †Chemistry and ‡Biology, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Mackenzie J
authors.initials: MJ
authors.lastname: Parker
authors.forename: Xuling
authors.initials: X
authors.lastname: Zhu
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D055162:Biocatalysis; D054477:Glutaredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008345:Manganese / Q000737:chemistry; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D054477:Glutaredoxins; D021122:Protein Subunits; D011994:Recombinant Proteins; D008345:Manganese; D013879:Thioredoxins; D012264:Ribonucleotide Reductases; D013880:Thioredoxin-Disulfide Reductase