Unveiling the Pathogenic Molecular Mechanisms of the Most Common Variant (p.K329E) in Medium-Chain Acyl-CoA Dehydrogenase Deficiency by in Vitro and in Silico Approaches

Medium-chain acyl-CoA dehydrogenasedeficiency ( MCADD) is the most common genetic disorderaffecting the mitochondrial fatty acid β-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramerassembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme’s activity. The most frequent mutation in MCADDresults from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein). Here, we combined in vitro and in silicoapproaches to assess the impact of the p.K329E mutation on the protein’s structure and function. Our in silicoresults demonstrated for the first time that the p.K329E mutation, despite lying at the dimer–dimer interface and being deeply buried inside the tetrameric core, seems to affect the tetramer surface, especially the β-domain that forms part of the catalytic pocket wall. Additionally, the molecular dynamics dataindicate a stronger impact of the mutation o...