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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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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.
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f5: 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.

Mentions: In addition to 2OG, the NMR assays revealed formation of succinate and another oxidation product, which on the basis of NMR and MS studies was assigned as succinic semialdehyde (SSA) by comparison with a synthetic standard (Fig. 4d, Supplementary Figs 10 and 11) and LC-MS assays (Supplementary Fig. 12). Fenton type chemistry is viable for α-hydroxyacid oxidation37; therefore, the possibility of H2O2-mediated 2HG oxidation was studied. It was found that 2HG oxidation is enabled by H2O2 to give 2OG, SSA and succinate, and that Fe(II) or Fe(III) are required for this transformation (Fig. 5, Supplementary Fig. 13). Interestingly, while 2HG oxidation is relatively slow under the conditions of the Fe(II)/L-ascorbate system (16–20 h or more), the H2O2-driven reaction is substantially faster (2OG formation was apparent by NMR after 1 h of incubation) (Supplementary Fig. 14). The observed ratio between 2OG and SSA was found to be dependent on the amount of H2O2 added (Supplementary Figs 15 and 16). To investigate whether SSA is the product of further oxidation of 2OG, 2OG oxidation under Fe(II)/L-ascorbate and Fe(II)/H2O2 conditions was also studied. 2OG was found to be oxidized to succinate, but not to detectable levels of SSA (Supplementary Figs 17 and 18), indicating that SSA formation occurs via an alternative 2HG oxidative pathway. Detectable SSA oxidation to succinate was not observed under these conditions (Supplementary Fig. 14). Therefore the 2HG oxidation reaction most likely proceeds via iron-complex formation. Overall, the results reveal that 2HG is oxidized to 2OG and SSA via (at least) two alternative oxidative pathways, and the ratio of the products depends on the amount of H2O2 present.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3959194&req=5

f5: 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.
Mentions: In addition to 2OG, the NMR assays revealed formation of succinate and another oxidation product, which on the basis of NMR and MS studies was assigned as succinic semialdehyde (SSA) by comparison with a synthetic standard (Fig. 4d, Supplementary Figs 10 and 11) and LC-MS assays (Supplementary Fig. 12). Fenton type chemistry is viable for α-hydroxyacid oxidation37; therefore, the possibility of H2O2-mediated 2HG oxidation was studied. It was found that 2HG oxidation is enabled by H2O2 to give 2OG, SSA and succinate, and that Fe(II) or Fe(III) are required for this transformation (Fig. 5, Supplementary Fig. 13). Interestingly, while 2HG oxidation is relatively slow under the conditions of the Fe(II)/L-ascorbate system (16–20 h or more), the H2O2-driven reaction is substantially faster (2OG formation was apparent by NMR after 1 h of incubation) (Supplementary Fig. 14). The observed ratio between 2OG and SSA was found to be dependent on the amount of H2O2 added (Supplementary Figs 15 and 16). To investigate whether SSA is the product of further oxidation of 2OG, 2OG oxidation under Fe(II)/L-ascorbate and Fe(II)/H2O2 conditions was also studied. 2OG was found to be oxidized to succinate, but not to detectable levels of SSA (Supplementary Figs 17 and 18), indicating that SSA formation occurs via an alternative 2HG oxidative pathway. Detectable SSA oxidation to succinate was not observed under these conditions (Supplementary Fig. 14). Therefore the 2HG oxidation reaction most likely proceeds via iron-complex formation. Overall, the results reveal that 2HG is oxidized to 2OG and SSA via (at least) two alternative oxidative pathways, and the ratio of the products depends on the amount of H2O2 present.

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