PMID- 27976856 OWN - NLM STAT- MEDLINE DCOM- 20170530 LR - 20170530 IS - 1520-4995 (Electronic) IS - 0006-2960 (Linking) VI - 55 IP - 51 DP - 2016 Dec 27 TI - 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. PG - 7086-7098 LID - 10.1021/acs.biochem.6b00759 [doi] AB - Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid beta-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 beta-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. FAU - Bonito, Catia A AU - Bonito CA AUID- ORCID: 0000-0003-3863-3033 AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Nunes, Joana AU - Nunes J AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Leandro, Joao AU - Leandro J AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Louro, Filipa AU - Louro F AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Leandro, Paula AU - Leandro P AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Ventura, Fatima V AU - Ventura FV AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. FAU - Guedes, Rita C AU - Guedes RC AD - Department of Biochemistry and Human Biology, section signMedicinal Chemistry, Research Institute for Medicines, iMed.ULisboa, double daggerMetabolism and Genetics Group, Research Institute for Medicines, iMed.ULisboa, and parallelDepartment of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, Universidade de Lisboa , Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. LA - eng PT - Journal Article DEP - 20161215 PL - United States TA - Biochemistry JT - Biochemistry JID - 0370623 RN - 3KX376GY7L (Glutamic Acid) RN - EC 1.3.8.7 (Acyl-CoA Dehydrogenase) RN - K3Z4F929H6 (Lysine) SB - IM MH - Acyl-CoA Dehydrogenase/chemistry/deficiency/*genetics MH - Biocatalysis MH - *Computer Simulation MH - Enzyme Stability MH - Glutamic Acid/genetics MH - Humans MH - Kinetics MH - Lipid Metabolism, Inborn Errors/enzymology/*genetics MH - Lysine/genetics MH - Models, Molecular MH - Motion MH - *Mutation, Missense MH - Principal Component Analysis MH - Protein Binding MH - Protein Domains MH - Protein Multimerization MH - Temperature EDAT- 2016/12/16 06:00 MHDA- 2017/05/31 06:00 CRDT- 2016/12/16 06:00 PHST- 2016/12/16 06:00 [pubmed] PHST- 2017/05/31 06:00 [medline] PHST- 2016/12/16 06:00 [entrez] AID - 10.1021/acs.biochem.6b00759 [doi] PST - ppublish SO - Biochemistry. 2016 Dec 27;55(51):7086-7098. doi: 10.1021/acs.biochem.6b00759. Epub 2016 Dec 15.