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Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.
Mackenzie J Parker1, Xuling Zhu, JoAnne Stubbe
1Departments of †Chemistry and ‡Biology, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Biochemistry
|January 10, 2014
Summary
Bacillus subtilis ribonucleotide reductase (RNR) activity was optimized by purifying its NrdF subunit and identifying endogenous reductants, TrxA and TrxB. This significantly enhanced enzyme function for potential structural studies.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Class Ib ribonucleotide reductase (RNR) from Bacillus subtilis was purified but exhibited low activity.
- The enzyme's tight subunit association suggested potential for crystallographic studies of an active complex.
Purpose of the Study:
- To optimize Bacillus subtilis RNR activity for structural and functional characterization.
- To isolate NrdF with a homogeneous cofactor and identify endogenous reductants.
Main Methods:
- Anion exchange chromatography for NrdF purification.
- Cloning, expression, and purification of TrxA, YosR, and TrxB as potential reductants.
- Enzyme activity assays, size exclusion chromatography, and analytical ultracentrifugation.
Main Results:
- Homogeneous Mn(III)2-Y· NrdF was isolated, containing 4 Mn and 1 Y·/β2.
- Endogenous reductants TrxA (thioredoxin) and TrxB (thioredoxin reductase) were identified and purified.
- Specific activity increased to ~1250 nmol min(-1) mg(-1) with optimized components.
Conclusions:
- Optimized conditions significantly enhanced Bacillus subtilis RNR activity.
- Characterization revealed monomeric NrdE and dimeric NrdF at physiological concentrations.
- The NrdE:NrdF complex exhibited a mixture of quaternary structures, differing from expectations.
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