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Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases.

Tarhonskaya H, Rydzik AM, Leung IK, Loik ND, Chan MC, Kawamura A, McCullagh JS, Claridge TD, Flashman E, Schofield CJ - Nat Commun (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.

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.

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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).
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f2: 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).

Mentions: To investigate the mechanism by which PHD activity is enabled by the presence of 2HG, MALDI-TOF-MS (matrix assisted laser desorption ionization-time of flight-mass spectrometry) based assays were performed using a peptide fragment of the HIF-1α C-terminal oxygen-dependent degradation domain (HIF-1α556–574, hereafter CODD) and recombinant PHD2181–426 (hereafter PHD2). In contrast to Koivunen et al.21, when substituting 300 μM 2OG for 5 mM 2HG in our standard steady-state assay conditions (4 μM PHD2, 200 μM CODD; Supplementary Table 1), we did not observe any CODD hydroxylation after short incubation times (up to 2 h, limit of detection: ~3% CODD hydroxylation). CODD hydroxylation was observed, however, with longer incubation times (20 h) (Fig. 2c,d) in the presence of both (R)- and (S)-2HG.


Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases.

Tarhonskaya H, Rydzik AM, Leung IK, Loik ND, Chan MC, Kawamura A, McCullagh JS, Claridge TD, Flashman E, Schofield CJ - Nat Commun (2014)

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).
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f2: 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).
Mentions: To investigate the mechanism by which PHD activity is enabled by the presence of 2HG, MALDI-TOF-MS (matrix assisted laser desorption ionization-time of flight-mass spectrometry) based assays were performed using a peptide fragment of the HIF-1α C-terminal oxygen-dependent degradation domain (HIF-1α556–574, hereafter CODD) and recombinant PHD2181–426 (hereafter PHD2). In contrast to Koivunen et al.21, when substituting 300 μM 2OG for 5 mM 2HG in our standard steady-state assay conditions (4 μM PHD2, 200 μM CODD; Supplementary Table 1), we did not observe any CODD hydroxylation after short incubation times (up to 2 h, limit of detection: ~3% CODD hydroxylation). CODD hydroxylation was observed, however, with longer incubation times (20 h) (Fig. 2c,d) in the presence of both (R)- and (S)-2HG.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.

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.

Show MeSH
Related in: MedlinePlus