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L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

Nature Chemical Biology volume 13, pages 494–500 (2017)Cite this article

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Abstract

The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D- R- or L- S- enantiomer, each of which inhibits α-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase (IDH) produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via 'promiscuous' reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

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Figure 1: LDH and MDH enzymes catalyze reduction of the alternative substrate αKG.
Figure 2: LDH and MDH selectively produce L-α-hydroxyacids, including L-2HG.
Figure 3: ACIDIC PH ENHANCES L-2HG production by LDH and MDH.
Figure 4: LDH exhibits pH-sensitive reduction of α-ketoacids with carboxylate tails.
Figure 5: Acidity-induced rate enhancement arises from preference of LDHA for the protonated form of αKG.
Figure 6: Acid-enhanced production of L-2HG stabilizes HIF-1α.

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Acknowledgements

We thank members of the Thompson laboratory for helpful discussions. We thank M. Isik, S. Hanson, and A. Rizzi from the Chodera laboratory for assistance. A.M.I. was supported by the NIH/NCI (K08 CA201483-01A1), the Leukemia & Lymphoma Society (Special Fellow Award 3356-16), the Burroughs Wellcome Fund (Career Award for Medical Scientists 1015584), the Conquer Cancer Foundation of ASCO, the Susan and Peter Solomon Divisional Genomics Program, and the Steven A. Greenberg Fund. The work was also supported, in part, by the Leukemia & Lymphoma Society Specialized Center of Research Program (7011-16), the Starr Cancer Consortium (I6-A616), and grants from the NIH, including R01 CA168802-02 and R01 CA177828-02 (C.B.T.), K99 CA191021-01A1 (C.C.-F.), and the Memorial Sloan Kettering Cancer Center Support Grant (NIH P30 CA008748). M.R.G. and S.S. received financial support from the National Science Foundation (MCB 1022208) and infrastructure support from the National Institute on Minority Health and Health Disparities (8G12MD007603-29).

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Authors and Affiliations

  1. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Andrew M Intlekofer, Bo Wang & Craig B Thompson

  2. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Andrew M Intlekofer

  3. The Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Hui Liu, Hardik Shah & Justin R Cross

  4. Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Carlos Carmona-Fontaine, Ariën S Rustenburg & John D Chodera

  5. Physics Department, City College of New York, New York, New York, USA

    Salah Salah & M R Gunner

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Contributions

A.M.I. and C.B.T. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. A.M.I. and B.W. performed all of the experiments. H.L., H.S., and J.R.C. assisted with liquid chromatography–mass spectrometry. C.C.-F. assisted with intracellular pH measurement. A.S.R., S.S., M.R.G., and J.D.C. performed the computational modeling. All authors read and approved the manuscript.

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Correspondence to Craig B Thompson.

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C.B.T. is a founder of Agios Pharmaceuticals and is a member of its scientific advisory board. He also serves on the board of directors of Merck and Charles River Laboratories. J.D.C. is a member of the scientific advisory board for Schrödinger, LLC.

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Intlekofer, A., Wang, B., Liu, H. et al. L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH. Nat Chem Biol 13, 494–500 (2017). https://doi.org/10.1038/nchembio.2307

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