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Tying down the arm in Bacillus dUTPase: structure and mechanism
Javier García-Nafría1, Jennifer Timm, Charlotte Harrison
1Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.
Summary
Bacillus subtilis dUTPases YncF and YosS exhibit unique structural features. Crystal structures reveal substrate-induced ordering, offering new insights into their catalytic mechanisms compared to other dUTPases.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Homotrimeric dUTPases possess three active sites formed by conserved motifs.
- The fifth motif, crucial for catalysis, resides in a flexible C-terminal arm.
- Bacillus subtilis dUTPases (YncF, YosS) show sequence and conformational differences from orthologues.
Purpose of the Study:
- To elucidate the structural basis of Bacillus subtilis dUTPase (YncF, YosS) function.
- To investigate substrate-induced conformational changes in these enzymes.
- To compare the catalytic mechanisms of B. subtilis dUTPases with their orthologues.
Main Methods:
- X-ray crystallography of YncF-dU-PPi-Mg(2+) and YosS-dUMP complexes.
- Limited proteolysis experiments.
- Analysis of enzyme structures in relation to catalytic reaction coordinate.
Main Results:
- dU-PPi-Mg(2+) induces ordering of the C-terminal arm and a unique loop (residues 18-26) in B. subtilis dUTPases.
- The YosS-dUMP complex suggests distinct stages of substrate release.
- Limited proteolysis identified ordered and hindered C-terminal cleavage states.
- dUpNHpp may not be an ideal substrate mimic for B. subtilis dUTPases.
Conclusions:
- Substrate binding triggers significant conformational changes in B. subtilis dUTPases.
- The study provides a dynamic view of dUTPase catalysis.
- Differences in substrate mimicry and enzyme structure offer insights into dUTPase evolution and mechanism.
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