Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis, regulated by nucleotide-binding domains.
• Aerobic RNRs are well-characterized, with deoxyadenosine triphosphate (dATP) inhibition mechanisms involving oligomerization.
• The dATP inhibition mechanism for anaerobic RNRs remains unknown, despite their distinct structural features like a stable glycyl radical domain (GRD).

Purpose of the Study:

• To elucidate the biochemical, biophysical, and structural basis of ATP and dATP binding to the anaerobic RNR from *Prevotella copri*.
• To understand the mechanism of dATP-dependent inhibition in anaerobic RNRs.

Main Methods:

• Biochemical assays to measure enzyme activity.
• Biophysical techniques to study protein dynamics and oligomerization.
• Cryo-electron microscopy (cryo-EM) for structural determination.

Main Results:

• ATP binding favors a dimeric state and active enzyme with an ordered GRD.
• dATP binding shifts the equilibrium to a tetrameric state, inactivates the enzyme, and increases GRD flexibility, preventing substrate binding.
• Cryo-EM structures reveal a regulatory network involving the GRD, substrate specificity site, and an active site flap, with dATP inducing conformational changes distant from the binding site.

Conclusions:

• dATP inhibits anaerobic *P. copri* NrdD by destabilizing the GRD and an active site flap, hindering substrate access and radical mobilization.
• This mechanism differs from aerobic RNRs, highlighting unique regulatory strategies in anaerobic enzymes.
• The findings provide crucial insights into the regulation of DNA synthesis precursors in anaerobic organisms.