Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.
Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.
复制标题
枯草芽孢杆菌 Ib 类核糖核苷酸还原酶：高活性和动态亚基相互作用。
DOI：
10.1021/bi401056e
复制
发表时间：
2014
期刊：
影响因子：
2.9
通讯作者：
Stubbe,JoAnne
中科院分区：
文献类型：
--
作者：
Parker,MackenzieJ;Zhu,Xuling;Stubbe,JoAnne
The class Ib ribonucleotide reductase (RNR) isolated fromBacillus subtiliswas 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–1mg–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested thatB. subtilisRNR 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 ofB. subtilisRNR 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 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–1mg–1using a 1:1 mixture of NrdE:MnIII2-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 MnIII2-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.
