Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases - PubMed (original) (raw)
Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases
Hanna Tarhonskaya et al. Nat Commun. 2014.
Free PMC article
Abstract
Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest.
Figures
Figure 1. The proposed catalytic mechanism for 2OG-dependent oxygenases.
(a) Binding of dioxygen, which occurs subsequently to enzyme–substrate complex formation, results in the oxidative decarboxylation of 2OG and generation of an Fe(IV)-oxo species that enables hydroxylation. (b) Structures of 2HG (2-hydroxyglutarate) and 2OG (2-oxoglutarate).
Figure 2. PHD2/2HG catalysis is enabled by reducing agents.
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).
Figure 3. 2HG conversion to 2OG is non-enzymatic.
(a,b) LC-MS chromatograms showing 2OG formation under non-enzymatic conditions in the presence of GSH (glutathione). (c,d) Amounts of 2OG formed with different reducing agents. Error bars represent s.d. of the mean of triplicate assays. The reaction mixture was incubated for 20 h (37 °C) and contained 5 mM (R)- or (S)-2HG, 50 μM Fe(II), reducing agent (from bottom to top in (a,b): no reducing agent; 0.5 mM; 4 mM; 10 mM reducing agent). All panels in one stack in (a,b) have the same range of ion intensity (0–6.5·105).
Figure 4. Non-enzymatic conversion of (S)- and (R)-2HG to 2OG.
(a) 1H NMR spectra showing conversion of (S)_-_2HG (2-hydroxyglutarate) to 2OG (2-oxoglutarate). The 1H spectra show the 2OG C
H
2COCO2H 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.
Figure 5. 1H NMR studies of (R)_-_2HG oxidation mediated by H2O2.
Reaction of (R)-2HG with H2O2 in a 90% water 10% D2O mixture ((a) 5 mM (R)_-_2HG, (b) 5 mM (R)_-_2HG, 50 μM Fe(II); (c) 5 mM (R)-2HG, 10 mM
L
-ascorbate; (d) 5 mM (R)_-_2HG, 5 mM H2O2, (e) 5 mM (R)_-_2HG, 50 μM Fe(II), 5 mM H2O2; (f) 5 mM (R)_-_2HG, 10 mM
L
-ascorbate, 5 mM H2O2; (g) 5 mM (R)_-_2HG, 50 μM Fe(II), 10 mM
L
-ascorbate; (h) 5 mM (R)_-_2HG, 50 μM Fe(II), 10 mM
L
-ascorbate, 5 mM H2O2). 2HG, 2-hydroxyglutarate; 2OG, 2-oxoglutarate; SA, succinic acid; SSA, succinic semialdehyde. Addition of both Fe(II)/
L
-ascorbate/H2O2 leads to impaired 2OG and SSA formation, likely due to competition between different oxidative pathways and/or further oxidation of 2OG and SSA.
Figure 6. Carnitine formation catalysed by BBOX1 in the presence of 2HG.
(a) Standards of (S)-2HG (2-hydroxyglutarate) and (e) (R)-2HG; (b) (S)-2HG or (f) (R)-2HG incubated with the assay mixture for 24 h prior to analysis; (c) (S)-2HG or (g) (R)-2HG incubated in the assay mixture with BBOX1 for 24 h prior to analysis; (d) (S)-2HG or (h) (R)-2HG incubated in the assay mixture for 24 h, then treated with BBOX1 and Fe(II) (1:1) and incubated for a further 2 h prior to analysis. The standard assay mixture contained 100 μM GBB (γ-butyrobetaine), 4 mM
L
-ascorbate, 50 μM Fe(II), 80 mM KCl in 50 mM Tris-d11 pH 7.5 containing 10% D2O. BBOX1 concentration was 100 nM. (S)-2HG and (R)-2HG were used at a final concentration of 5 mM.
Figure 7. Metabolic pathways of (R)- and (S)-2HG.
Red arrows represent non-enzymatic transformations observed in this study, black arrows represent identified metabolic pathways.
L
-maIDH,
L
-malate dehydrogenase;
L
-2-HGDH,
L
-2-hydroxyglutarate dehydrogenase; IDH2, isocitrate dehydrogenase 2;
D
-2HGDH,
D
-2-hydroxyglutarate dehydrogenase; HOT, hydroxyacid-oxoacid transhydrogenase enzyme; GBH, γ-hydroxybutyrate; SSA reductase, succinic semialdehyde reductase enzyme; SSADH, succinic semialdehyde dehydrogenase; GABA, γ-aminobutyric acid.
References
- Matsunaga H. et al. IDH1 and IDH2 have critical roles in 2-hydroxyglutarate production in D-2-hydroxyglutarate dehydrogenase depleted cells. Biochem. Biophys. Res. Commun. 423, 553–556 (2012). - PubMed
- Ichimura K. Molecular pathogenesis of IDH1/2 mutations in gliomas. Brain Tumor Pathol. 29, 131–139 (2012). - PubMed
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