Figure 2: PHD2/2HG catalysis is enabled by reducing agents.
From: Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases
Samples containing 4 μM PHD2 (prolyl hydroxylase domain 2), 200 μM C-terminal oxygen dependent degradation domain (CODD) peptide, 5 mM (R)- or (S)-2HG, 50 μM Fe(II), 0/0.5/4/10 mM L-ascorbate in Hepes 50 mM pH 7.5 were incubated for 20 h (37 °C) and then analysed by MALDI-TOF-MS or subjected to amino acid analysis. Error bars represent s.d. of the mean of triplicate assays. 2OG (2-oxoglutarate) control incubations contained 300 μM 2OG instead of 2HG. (a) The PHD2-catalysed reaction. (b) Typical MALDI-TOF-MS spectra of CODD-OH (upper) and CODD (lower). (c) Dependence of PHD2/(R)-2HG-catalysed CODD hydroxylation on L-ascorbate and GSH (glutathione). (d) Dependence of PHD2/(S)-2HG-catalysed CODD hydroxylation reaction on L-ascorbate and GSH. (e,f) Amino acid analysis results (A: 2HG, Fe(II), L-ascorbate; B: CODD, 2HG, Fe(II), L-ascorbate; C: PHD2, 2HG, Fe(II), L-ascorbate; D: PHD2, CODD, 2HG, Fe(II), L-ascorbate; E: standard containing trans-4-hydroxyproline amino acid).
