Circular diochroism (CD) spectra of the wild-type SmIDH and four mutants, S102T, S102G, S102A and S102Y.

<p>The CD was measured and the molar ellipticity was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058918#s2" target="_blank">Materials and methods</a>.</p

Enzyme purity, western blot and molecular mass analysis of SmIDH.

<p>(A) SDS-PAGE analysis of the expression and purification of SmIDH. Analysis was performed on 12% polyacrylamide gel. M, protein markers; lane 1, crude extracts of cells harboring pWT grown in MD medium without glutamate. lane 2, purified protein. (B) Western blot analysis using anti-6×His antibody as probe. (C) Molecular mass determination by gel filtration chromatography. The flow rate was 0.5 ml/min and the proteins in the fractions were monitored at 280 nm. Inside is the standard curve for molecular mass. SmIDH was represented as a dark circle (•). Standard proteins were represented by open circles (○): 1, Ovalbumin (44 kDa); 2, Conalbumin (75 kDa); 3, Aldolase (158 kDa); 4, Ferritin (440 kDa); 5, Thyroglobulin (669 kDa). <i>V</i>_e of SmIDH is 14.4 ml.</p

Kinetic analysis of the recombinant SmIDH.

<p>The kinetic parameters of the recombinant SmIDH were determined by measuring its enzyme activity at various isocitrate or NAD⁺ concentrations with the other substrate at saturating concentrations. Enzymatic activity was assessed by monitoring the increase of NADH. The SmIDH <i>K</i>_m for NAD⁺ (A) and isocitrate (B) were calculated as 154 μM and 75 μM, respectively, by averaging values from triplicate experiments.</p

Structure-based protein sequence alignment of SmIDH with other dimeric IDHs.

<p>High-resolution crystal structures of <i>E. coli</i> NADP-IDH (EcIDH, 9ICD), <i>B. subtilis</i> NADP-IDH (BsIDH, 1HQS) and <i>A. thiooxidans</i> NAD-IDH (AtIDH, 2D4V) were downloaded from the PDB database. SmIDH structure was generated using the SWISS-MODEL modeling server, using AtIDH as a template structure. Invariant residues are highlighted by shaded blue boxes and conserved residues by open blue boxes. The conserved residues involved in the cofactor binding (▴) and substrate binding (*) are indicated. The phosphorylation site was represented by ★. The phosphorylation loop and the insert region were highlighted by shaded orange boxes. The figure was made with ESPript 2.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058918#pone.0058918-Gouet1" target="_blank">[36]</a>.</p

Effects of metal ions on the activity of recombinant SmIDH ^a.

a<p>The values indicate the means of at least three independent measurements.</p

The residual activities of the four Ser102 mutants as compared to the wild-type SmIDH.

<p>The residual activities of the four Ser102 mutants as compared to the wild-type SmIDH.</p

Construction of SmIDH mutants.

a<p>Dashes indicate the same amino acid residues as SmIDH.</p>b<p>Only sense primers are shown. Underlines indicate mutated regions.</p

Comparison of the kinetic parameters for the α-KG reduction activity of the mutant IDHs.

<p>Comparison of the kinetic parameters for the α-KG reduction activity of the mutant IDHs.</p

Structure-based protein sequence alignment of ScIDH1, YlIDH and HcIDH.

<p>The conserved amino acid residues are shaded. The secondary structure components, j.e, α-helices and β-sheets, of HcIDH and ScIDH1 are also shown. The conserved amino acid residues involved in substrate binding (★) and cofactor binding (▴) are indicated. This figure was generated using ESPript 2.2.</p

Primers used in this study.

a<p>“-S” and “-As”: indicate the sense (-S) and antisense (-As) primers of the corresponding genes.</p><p>b “-f” and “-r”: indicate the forward (-f) and reverse (-r) primers used in the site-directed mutagenesis.</p><p>Underlined bases indicate the mutant sites.</p><p>Primers used in this study.</p