Figure 1.
Coomassie-stained SDS-PAGE gel of purified HsDH recombinant protein and its five clinically interesting patient variants under reducing conditions.
The first line represents Low molecular weight protein standard (Bio-Rad) and lines 2–7 protein samples purified by Ni-NTA and Superdex 200 gel filtration columns as follows: 2) wild type HsDH, 3) T15A variant, 4) N158D variant, 5) E232K variant, 6) R248C variant and 7) W249G variant. The molecular masses of all the recombinant protein monomers (∼36 kDa) correspond to the monomer mass (36.14 kDa) estimated from the aa sequence of the wild type HsDH [29].
Table 1.
Features of native recombinant HsDH protein and its five clinically interesting patient mutation variants.
Figure 2.
Growth of the BY4741 Δfox2 cells on oleic acid transformed with pYE352::ScMFE-2, pYE352::CTA1, pYE352::HsMFE-2, and its five clinically interesting patient variants.
The dilution series were done on YPD (0.2% glucose)- oleic acid (0.125%) plates and the BY4741 Δfox2 cells were grown at +30°C for one week indicated by: (1) BY4741 Δfox2+ pYE352::HsMFE-2, (2) BY4741 Δfox2+ pYE352::ScMFE-2, (3) BY4741 Δfox2 strain, (4) BY4741 Δfox2+ pYE352::CTA1, (5) BY4741 Δfox2+ pYE352::HsMFE-2(T15A), (6) BY4741 Δfox2+ pYE352::HsMFE-2(N158D), (7) BY4741 Δfox2+ pYE352::HsMFE-2(E232K), (8) BY4741 Δfox2+ pYE352::HsMFE-2(R248C), (9) BY4741 Δfox2+ pYE352::HsMFE-2(W249G). When the yeast is able to utilize the oleic acid as a sole source of carbon, there will appear a clear zone around (samples 1, 2, 5, 6, 7, 8 & 9), but if the functional fox2 gene is missing the environment remains opaque (samples 3 & 4).
Table 2.
The hydrogen bonds analyzed in MD simulations.
Figure 3.
Five biologically interesting variations located in the dehydrogenase dimer of human MFE-2.
The two dehydrogenase monomers in the middle of the figure are colored in green and blue, amino acids T15, N158, E232, R248 and W249 are shaded grey and shown in stick presentations, NAD+ are colored in yellow and shown also in stick presentation. The rectangles indicate parts of the structure that are represented in larger details in small figures. The figures were done using the program PyMol (Schrödinger) and human 3R-hydroxyacyl-CoA dehydrogenase structure (PDB ID 1ZBQ; [7]).
Figure 4.
Hydrogen bond distance evolutions correlated with the variations.
The two dehydrogenase monomers are shown as surface presentation; monomer A is colored in green and monomer B in blue. For both monomers the larger (upper) part in panel A is the Rossmann fold core domain and the smaller (lower) part is the C-terminal subdomain. Variation sites are color coded with the fluctuation analysis as follows: N158D (green), E232K (violet), R248C (yellow) and W249G (light brown). Residues found essential for discussion on the D-BP deficiency mechanism are colored as follows: amino acids belonging to catalytic triad (S151, T164 and K168) in magenta, amino acids locating at the tip of the cavity leading to the active site (I180, Y156 [36] and I288) in purple blue and amino acids important for the substrate binding (E250 and R251) in orange [36]. NAD+ is colored in yellow and presented in stick presentation. (A) The front side of both monomers. The groove for the substrate binding begins from the crossing area of C-terminal subdomains and continues to the cavity leading to the active site. Four different areas in which the variations cause structural fluctuation compared to native protein are marked with ellipses and linked to the fluctuation figures. In the fluctuation figures each rectangle shows the evolution of the distance (in nm; y-axis) of a specific hydrogen bond (as indicated) for 50 ns (x-axis). On each four fluctuation figures the leftmost rectangle (in black) is the native protein, while the other rectangles show the distance evolution of the indicated hydrogen bond in protein variants as follows: N158D (top left and bottom right in green), W249G (top left and right in light brown) and E232K (bottom left in violet). Representative surface-exposed hydrogen bonds were chosen for the figure; a complete list of hydrogen bonds, which were found fluctuating, is presented in table 2. (B) The substrate binding groove and cavity from above. The figures were done using the program PyMol (Schrödinger) and human 3R-hydroxyacyl-CoA dehydrogenase structure (PDB ID 1ZBQ).