Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions
Name
Stubbe_Bacillus subtilis.pdf
Size
1.09 MB
Format
Adobe PDF
Checksum (MD5)
00131c341d8065e3878e83abfa43d913
Author(s)
Zhu, Xuling
Stubbe, JoAnne
Parker, Mackenzie James
Date Issued
January 2014
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
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.
Version
Author's final manuscript
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[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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1021/bi401056e
