title: 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.
abstract: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid β-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled 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 MCADD results 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 silico approaches to assess the impact of the p.K329E mutation on the protein's structure and function. Our in silico results 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 data indicate a stronger impact of the mutation on the protein's motions in dimer A/B, while dimer C/D remains similar to the wild type. For dimer A/B, severe disruptions in the architecture of the pockets and in the FAD and octanoyl-CoA binding affinities were also observed. The presence of unaffected pockets (C/D) in the in silico studies may explain the decreased enzymatic activity determined for the variant protein (46% residual activity). Moreover, the in silico structural changes observed for the p.K329E variant protein provide an explanation for the structural instability observed experimentally, namely, the disturbed oligomeric profile, thermal stability, and conformational flexibility, with respect to the wild-type.
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Cátia A
authors.identifier: http://orcid.org/0000-0003-3863-3033
authors.initials: CA
authors.lastname: Bonito
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Nunes
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: João
authors.initials: J
authors.lastname: Leandro
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Filipa
authors.initials: F
authors.lastname: Louro
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Leandro
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Fátima V
authors.initials: FV
authors.lastname: Ventura
authors.affiliation: Department of Biochemistry and Human Biology, §Medicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, ‡Metabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and ∥Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: Rita C
authors.initials: RC
authors.lastname: Guedes
mesh_terms: D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*; D055162:Biocatalysis; D003198:Computer Simulation; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008052:Lipid Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D009038:Motion; D020125:Mutation, Missense; D025341:Principal Component Analysis; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D013696:Temperature
chemical_list: D018698:Glutamic Acid; D042964:Acyl-CoA Dehydrogenase; D008239:Lysine