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

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• 作者：Mackenzie J. Parker ; Xuling Zhu ; JoAnne Stubbe
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：February 4, 2014
• 年：2014
• 卷：53
• 期：4
• 页码：766-776
• 全文大小：408K
• ISSN：1520-4995

文摘

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (伪) and NrdF (尾) subunits and determined to have a dimanganic-tyrosyl radical (Mn^III₂-Y路) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn^III₂-Y路 NrdF using dithiothreitol as the reductant, however, was low (160 nmol min^鈥? mg^鈥?). 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 鈥渁ctive鈥?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^III₂-Y路 NrdF, yielding enzyme containing 4 Mn and 1 Y路/尾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^鈥? mg^鈥? using a 1:1 mixture of NrdE:Mn^III₂-Y路 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 渭M), NrdE is a monomer (伪) and Mn^III₂-Y路 NrdF is a dimer (尾₂). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.