Glyoxylate reductase/hydroxypyruvate reductase regulates the free d‐aspartate level in mammalian cells

Multiple d‐amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d‐amino acids identified in mammals, d‐aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d‐aspartate. In particular, d‐aspartate is degraded by d‐aspartate oxidase (DDO), a peroxisome‐localized enzyme that catalyzes the oxidative deamination of d‐aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d‐aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm‐localized DDO; this cell line cannot survive in the presence of high concentrations of d‐aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d‐aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d‐aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d‐aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome‐localized DDO on d‐ and l‐aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells. Effects of expression of d‐Asp oxidase (DDO) and glyoxylate reductase/hydroxypyruvate reductase (GRHPR) on accumulation of d‐Asp in 293 cells and their culture medium. 293 cells stably transfected with plasmids encoding human DDO (293‐hDDO‐1), human GRHPR (293‐hGRHPR‐7), human DDO and human GRHPR (293‐hDDO‐hGRHPR‐4), or empty vector (293/NEO) were seeded in six‐well plates. The following day, the medium was replaced with a fresh medium containing 100 µM d‐Asp, the cells were cultured for another 2 days, and the d‐Asp contents in the cells (A) and culture media (B), as well as the total content (cells plus medium) (C), were determined.