RESUMO
Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid ß-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditiselegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L.
Assuntos
17-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/química , Oxirredutases do Álcool/química , Proteínas de Caenorhabditis elegans/química , Carbono-Oxigênio Liases/química , Proteínas de Drosophila/química , Enoil-CoA Hidratase/química , Hidroliases/química , Modelos Moleculares , Complexos Multienzimáticos/química , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/metabolismo , Proteínas de Transporte/química , Dimerização , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteína Multifuncional do Peroxissomo-2 , Peroxissomos/enzimologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Solubilidade , Especificidade da Espécie , Síncrotrons , Difração de Raios XRESUMO
Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m), V(max) and k(cat)) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.
