Figure 4: Non-enzymatic conversion of (S)- and (R)-2HG to 2OG.
From: Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases
(a) 1H NMR spectra showing conversion of (S)-2HG (2-hydroxyglutarate) to 2OG (2-oxoglutarate). The 1H spectra show the 2OG CH2COCO2H resonance. Conditions (from top to bottom): 50 μM Fe(II) and 10 mM L-ascorbate; 5 mM (S)-2HG and 50 μM Fe(II); 5 mM (S)-2HG and 10 mM L-ascorbate; 5 mM (S)-2HG; 5 mM (S)-2HG, 50 μM Fe(II) and 10 mM L-ascorbate. Reaction mixtures were incubated for 20 h at 25 °C. All solutions were buffered with 50 mM Tris-d11 (pH 7.5) and 0.02% NaN3 in 90% H2O and 10% D2O. (b) Time course of non-enzymatic 2OG production as analysed by 1H NMR. Conditions: 5 mM (S)-2HG, 5 μM Fe(II), 2 mM L-ascorbate. (c) Succinate and 2OG production (total concentration) in the assay mixtures containing (R)-2HG. Conditions: 5 mM (R)-2HG, 5 μM Fe(II), 2 mM L-ascorbate, 4 μM PHD2 (prolyl hydroxylase domain 2), 128 μM CODD (C-terminal oxygen dependent degradation domain). Reaction mixtures were incubated for 20 h at 37 °C, PHD2 was precipitated by incubation in boiling water for 30 s before NMR analyses. All solutions were buffered with 50 mM Tris-d11 (pH 7.5) and 0.02% NaN3 in 90% H2O and 10% D2O. Error bars represent s.d. of the mean of triplicate assays. (d) Scheme for 2HG conversion to 2OG. SA, succinic acid; SSA, succinic semialdehyde.
