ABSTRACT
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.
Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , 3-Hydroxyacyl CoA Dehydrogenases/chemistry , Alcohol Oxidoreductases/chemistry , Caenorhabditis elegans Proteins/chemistry , Carbon-Oxygen Lyases/chemistry , Drosophila Proteins/chemistry , Enoyl-CoA Hydratase/chemistry , Hydro-Lyases/chemistry , Models, Molecular , Multienzyme Complexes/chemistry , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/genetics , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/metabolism , Carrier Proteins/chemistry , Dimerization , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Enoyl-CoA Hydratase/genetics , Enoyl-CoA Hydratase/metabolism , Humans , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peroxisomal Multifunctional Protein-2 , Peroxisomes/enzymology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scattering, Small Angle , Solubility , Species Specificity , Synchrotrons , X-Ray DiffractionABSTRACT
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.
