Cryo-EM Structure of Recombinantly Expressed hUGDH Unveils a Hidden, Alternative Allosteric Inhibitor

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Human UDP-glucose dehydrogenase (hUGDH) is crucial for drug metabolism.
• hUGDH exists in equilibrium between active (E) and inactive (EΩ) states, influenced by allosteric inhibitors like UDP-xylose (UDP-Xyl).
• Previous studies presented conflicting evidence regarding the preferred state of unliganded hUGDH.

Purpose of the Study:

• To resolve the discrepancy in the allosteric mechanism of hUGDH.
• To investigate the role of UDP-4-keto-xylose (UX4O) in hUGDH conformational states.
• To determine the physiological relevance of UX4O as an inhibitor.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to visualize hUGDH structures.
• Biochemical assays to analyze enzyme kinetics and inhibition.
• Purification of recombinant hUGDH from Escherichia coli.

Main Results:

• Recombinant hUGDH copurified with UX4O, stabilizing the inactive EΩ state.
• Removal of UX4O shifted the hUGDH ensemble towards the active E state.
• Progress curve analysis confirmed the absence of a lag for unliganded hUGDH after UX4O removal.
• hUGDH exhibited similar binding affinities for UDP-Xyl and UX4O.

Conclusions:

• UX4O is identified as a key factor influencing hUGDH conformational states.
• The findings support the shift of unliganded hUGDH towards the active state.
• UX4O is proposed as a potential physiological inhibitor for bacterial UDP-glucose dehydrogenases.