Medium-chain acyl CoA dehydrogenase: evidence for phosphorylation

Area of Science:

• Biochemistry
• Mitochondrial Metabolism
• Enzyme Post-Translational Modifications

Background:

• Medium chain acyl-CoA dehydrogenase (MCADH) is a key mitochondrial enzyme involved in fatty acid oxidation.
• Post-translational modifications, such as phosphorylation, can regulate enzyme function and stability.
• The precise phosphorylation status of pig kidney MCADH (pkMCADH) and its functional implications are not fully understood.

Purpose of the Study:

• To investigate the phosphorylation state of mature pkMCADH.
• To determine the functional consequences of identified phosphorylation on MCADH activity and solubility.
• To compare the phosphorylation of native pkMCADH with recombinant human MCADH.

Main Methods:

• 31P-NMR spectroscopy to identify phosphate-containing groups.
• Chemical analysis to quantify phosphate content.
• Enzyme activity assays using artificial and natural electron acceptors.
• Protein solubility measurements.
• Enzymatic dephosphorylation using phosphatase and subsequent dialysis.

Main Results:

• Mature pkMCADH contains three phosphate groups: two associated with the FAD cofactor and one additional covalently attached phosphomonoester.
• Recombinant human MCADH and reconstituted pkMCADH holoenzyme contain only the two FAD-associated phosphates.
• The covalently bound phosphate is removable by phosphatase treatment.
• Dephosphorylation does not affect MCADH catalytic activity but significantly decreases protein solubility by five-fold.

Conclusions:

• Mature pkMCADH possesses a unique covalent phosphorylation distinct from its FAD cofactor.
• This covalent phosphorylation plays a crucial role in maintaining MCADH protein solubility.
• While not impacting catalytic efficiency, phosphorylation influences MCADH stability and potentially its cellular localization or interactions.