Bacillus subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

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 (MnIII2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted MnIII2-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 MnIII2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β2. Goal two was...