Xenbase Image ID: 127245

Figure 1. Crystal structure, stability and activity of the HSD10 homotetramer and mitochondrial morphology in fibroblasts from MHBDD patientsCrystal structure of HSD10. (A) Diagram of the homotetramer. A NADH cofactor is bound to one monomer. (B) Diagram of a HSD10 dimer with the location of the mutations. The active centre is marked by the substrate in red.C. Enzyme activity of HSD10 over time. Kcat/KM of HSD10 WT and mutations measured in a 30 min interval; substrate 2-methyl-3-hydroxybutyryl-CoA.D. Stability of HSD10 WT and mutations. Mean value of Tm determined by differential scanning fluorimetry (DSF)-experiments and standard deviation are shown. Black column: no cofactor/substrate, grey column: cofactor NAD+, white column: cofactor NADH (* indicates significant cofactor binding).E, F. Enzyme activity of HSD10 with different substrates. (E) Kcat/KM of HSD10 mutations with hydroxybutyryl-CoA as substrate (WT enzyme is taken as 100%). (F) Kcat/KM of HSD10 mutations with 2-methyl-3-hydroxybutyryl-CoA as substrate in relativity to the WT enzyme.G–J. Mitochondrial staining in patient fibroblasts. 300 nM Mitotracker Green FM were used on cells fixed with 3.7% formaldehyde on coverslips and mitochondria were visualized on a Perkin Elmer spinning disc confocal ERS-FRET on Nikon TE2000 inverted microscope. (G) Control, (H) R130C, (I) D86G, (J) Q165H.K. Fibroblasts were sectioned for electron microscopy. Pictures of 10–43 random systematically chosen visual fields were taken in a magnification of 11.5 × 103, scale bars: 100 nm. Mitochondria were classified into three groups (1—dense, dark; 2—loosely packed; 3—depleted cristae) with group 3 not only being morphologically distinct but also characterized by smaller mitochondria. Total numbers per sample and an overview of the cells are given in Supporting Information Fig 3. ** Indicate significance at p < 0.0001, * gives significance at p = 0.0366–0.0857 adjusted for multiple comparisons within the experiment compared to control fibroblasts. Normal human dermal fibroblasts served as controls. Image published in: Rauschenberger K et al. (2010) Image downloaded from an Open Access article in PubMed Central. Copyright © 2010 EMBO Molecular Medicine Larger Image Printer Friendly View

Return to previous page