Figure 1.
Normal and neomorphic reactions catalyzed by wild-type and mutant IDH.
The normal reaction converts isocitrate to α-ketoglutarate and CO2 accompanying with NADP+ reduction, while the neomorphic reaction transforms α-ketoglutarate to 2-hydroxyglutarate in an NADPH dependent manner.
Table 1.
Primers used in this study.
Figure 2.
Structure-based protein sequence alignment of ScIDH1, YlIDH and HcIDH.
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.
Figure 3.
SDS-PAGE analysis of the expression and purification of wild-type and mutant YlIDH, ScIDH1 and HcIDH.
Analysis was performed using a 12% polyacrylamide gel. M, protein molecular weight markers; lane 1 to 4, purified proteins of YlIDH, YlIDH R141H, YlIDH R141A and YlIDH R141E, respectively; lane 5 to 8, purified proteins of ScIDH, ScIDH R148H, ScIDH R148A and ScIDH R148E, respectively; lane 9 to 12, purified proteins of HcIDH, HcIDH R132H, HcIDH R132A and HcIDH R132E, respectively.
Figure 4.
Circular dichroism (CD) spectra analysis of intact and mutant YlIDH, ScIDH1 and HcIDH.
The CD was measured, and the molar ellipticity was calculated as described in the Materials and Methods. (A) The molar ellipticities of HcIDH (▪), HcIDH R148H (•), HcIDH R132A (▴) and HcIDH R132E (▾) from 195 to 260 nm; (B) the molar ellipticities of ScIDH1 (▪), ScIDH1 R148H (•), ScIDH1 R148A (▴) and ScIDH1 R148E (▾) from 195 to 260 nm; (C) the molar ellipticities of YlIDH (▪), YlIDH R141H (•), YlIDH R141A (▴) and YlIDH R148E (▾) from 195 to 260 nm.
Table 2.
Comparison of the kinetic parameters of the isocitrate oxidation activity of the wild-type and mutant IDH enzymes.
Figure 5.
Normal and neomorphic reaction analysis of the wild-type and mutant YlIDH and ScIDH1.
(A) The oxidation activity was determined using the standard assay conditions. The oxidation reaction was initiated by adding the appropriate amount of enzymes. The increasing of NADPH catalyzed by YlIDH (▪), YlIDH R141H (□), ScIDH1 (•) and ScIDH1 R148H (○), respectively. (B) The reduction of α-KG was determined using standard assay conditions. The enzymatic reaction was initiated by adding an appropriate amount of enzyme. The decrease of NADPH catalyzed by YlIDH (▪), YlIDH R141H (□), ScIDH1 (•) and ScIDH1 R148H (○), respectively.
Table 3.
Comparison of the kinetic parameters for the α-KG reduction activity of the mutant IDHs.
Figure 6.
GC/TOF-MS identification of the 2-HG, ICT and α-KG standards.
The retention times of the 2-HG, ICT and α-KG standards in the gas chromatogram were 11.94 min (A), 17.44 min (B) and 12.03 min (C), respectively. (D), (E) and (F) show the mass spectrogram identifications of 2-HG, ICT and α-KG, respectively.
Figure 7.
GC/TOF-MS identification of the products generated by YlIDH R141H and ScIDH1 R148H catalysis.
(A) and (D) Gas chromatograms of the products generated by YlIDH R141Hand ScIDH1 R148H catalysis, respectively. (B) and (E) Mass spectrogram identifications of the 11.94-min peak in (A) and 11.91-minpeak in (D), respectively.(C) and (F) Mass spectrogram identifications of the 17.43-min peak in (A) and (D), respectively.