Features of native recombinant <i>Hs</i>DH protein and its five clinically interesting patient mutation variants.

<p>The activities of human dehydrogenase and its variants were measured by detecting spectrophotometrically the formation of magnesium complex of 3R-hydroxydecanoyl-CoA at 303 nm from the 2E-decenoyl-CoA substrate in concentration range from 0.5 to 30 µM. Kinetic parameters are calculated by using GraFit 5.0 program (Erithacus Software). Main features characterizing the observed structural changes in the variant proteins and the patients are also given (and discussed in more details in the text).</p><p>n.d. = not determined (recording reliable and comparable data would have necessitated using a large surplus of NAD⁺).</p

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

<div><p>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 <em>in vivo</em> in yeast and <em>in vitro</em> enzyme kinetic and stability determinants as well as <em>in silico</em> 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.</p> </div

The hydrogen bonds analyzed in MD simulations.

<p>In total 18 hydrogen bonds, which were found to either be stable in the native protein or to form when the variation was present, were analyzed. The symbol (+) indicates the stable existence of the hydrogen bond and the symbol (–) clear fluctuation.</p

Five biologically interesting variations located in the dehydrogenase dimer of human MFE-2.

<p>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; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Lukacik1" target="_blank">[7]</a>).</p

Growth of the BY4741 <i>Δfox2</i> cells on oleic acid transformed with pYE352::<i>ScMFE-2,</i> pYE352::<i>CTA1</i>, pYE352::<i>HsMFE-2</i>, and its five clinically interesting patient variants.

<p>The dilution series were done on YPD (0.2% glucose)- oleic acid (0.125%) plates and the BY4741 <i>Δfox2</i> cells were grown at +30°C for one week indicated by: (1) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>, (2) BY4741 <i>Δfox2</i>+ pYE352::<i>ScMFE-2</i>, (3) BY4741 <i>Δfox2</i> strain, (4) BY4741 <i>Δfox2</i>+ pYE352::<i>CTA1</i>, (5) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(T15A), (6) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(N158D), (7) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(E232K), (8) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(R248C), (9) BY4741 <i>Δfox2</i>+ pYE352::<i>HsMFE-2</i>(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 <i>fox2</i> gene is missing the environment remains opaque (samples 3 & 4).</p

Coomassie-stained SDS-PAGE gel of purified <i>Hs</i>DH recombinant protein and its five clinically interesting patient variants under reducing conditions.

<p>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 <i>Hs</i>DH, 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 <i>Hs</i>DH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053688#pone.0053688-Gasteiger1" target="_blank">[29]</a>.</p