Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions
Author(s)Zhu, Xuling; Stubbe, JoAnne; Parker, Mackenzie James
MetadataShow full item record
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[superscript III][subscript 2]-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn[superscript III][subscript 2]-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min[superscript –1] mg[superscript –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[superscript III][subscript 2]-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·[over β [subscript 2]]. 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[superscript –1] mg[superscript –1] using a 1:1 mixture of NrdE:Mn[superscript III][subscript 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[superscript III][subscript 2]-Y· NrdF is a dimer (β[subscript 2]). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.
DepartmentMassachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
American Chemical Society (ACS)
Parker, Mackenzie J., Xuling Zhu, and JoAnne Stubbe. “ Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions .” Biochemistry 53, no. 4 (February 4, 2014): 766–776.
Author's final manuscript