Domain structure and function of 10-formyltetrahydrofolate dehydrogenase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• 10-Formyltetrahydrofolate dehydrogenase (10-formyl-THF dehydrogenase) catalyzes NADP(+)-dependent oxidation of 10-formyltetrahydrofolate.
• Previous research indicated separate binding sites for dehydrogenase and hydrolase activities within the enzyme's three domains.
• The enzyme exhibits high affinity for its tetrahydrofolate product.

Purpose of the Study:

• To investigate the domain structure and functional roles of 10-formyl-THF dehydrogenase from rabbit liver.
• To elucidate the specific activities associated with each domain of the enzyme.
• To characterize ligand binding properties of the native enzyme.

Main Methods:

• Purification of 10-formyl-THF dehydrogenase from rabbit liver.
• Differential scanning calorimetry to assess thermal stability and domain structure.
• Limited proteolytic digestion to identify functional domains.
• Kinetic analysis and ligand binding studies (fluorescence, isothermal titration calorimetry).

Main Results:

• Differential scanning calorimetry identified two independently folded domains.
• Proteolytic cleavage separated the domains, abolishing dehydrogenase activity.
• The N-terminal domain exhibited NADP(+)-independent 10-formyltetrahydrofolate hydrolase activity.
• The C-terminal domain demonstrated NADP(+)-dependent aldehyde dehydrogenase activity.
• Native enzyme binds one tetrahydrofolate and two NADP+ molecules per tetramer.

Conclusions:

• 10-Formyl-THF dehydrogenase is a two-domain homotetrameric enzyme.
• Each domain possesses distinct catalytic activities (hydrolase and aldehyde dehydrogenase).
• Domain separation inactivates the dehydrogenase function, highlighting the importance of domain association.