The purpose of this study was to identify the biochemical and genetic defect in L-2-hydroxyglutaric aciduria, a neurometabolic disorder characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid. Evidence is provided for the existence in rat tissues of a FAD-dependent enzyme catalyzing specifically the oxidation of L-2-hydroxyglutarate to ␣-ketoglutarate. This enzyme is mainly expressed in liver and kidney but also at lower levels in heart, brain, and other tissues. Subcellular fractionation indicates that the liver enzyme is present in mitochondria, where it is bound to membranes. Based on this information, a database search led to the identification of a gene encoding a human hypothetical protein homologous to bacterial FAD-dependent malate dehydrogenases and targeted to mitochondria. The gene encoding this protein, present on chromosome 14q22.1, was found to be in a region homozygous in patients with L-2-hydroxyglutaric aciduria from two consanguineous families. Three mutations that replaced a highly conserved residue (Lys-71-Glu and Glu-176-Asp) or removed exon 9 were identified in homozygous state in patients from three distinct families and were found to cosegregate with the disease. It is concluded that L-2-hydroxyglutarate is normally metabolized to ␣-ketoglutarate in mammalian tissues and that L-2-hydroxyglutaric aciduria is caused by mutations in the gene that most likely encodes L-2-hydroxyglutarate dehydrogenase. The pathological findings observed in this metabolic disorder must therefore be due to a toxic effect of L-2-hydroxyglutarate on the central nervous system. inborn error of metabolism ͉ leukoencephalopathy ͉ ataxia A s a neurometabolic disorder, L-2-hydroxyglutaric aciduria is characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in plasma, cerebrospinal fluid, and urine (1-3). Clinically, the affection is characterized by progressive ataxia, mental deficiency with subcortical leukoencephalopathy, and cerebellar atrophy. As of 1996, Ͼ40 patients were known worldwide (3). The disease appears to be recessively transmitted, but the enzymatic defect leading to this condition is presently unknown, largely because of poor knowledge on the origin and fate of L-2-hydroxyglutarate. A NAD-dependent dehydrogenase acting on this compound has been reported to be present in liver (4). However, this enzyme displays a very high K m for L-2-hydroxyglutarate (Ϸ10 mM), making it unsuitable to consume this substrate at physiologically relevant concentrations (probably of the order of 1 M). Furthermore, it is not deficient in patients with L-2-hydrox yglutaric aciduria (5). L-2-hydroxyglutarate dehydrogenase activity transferring its electrons to pyocyanin was partially characterized Ϸ70 years ago by Weil-Malherbe (6), who found it to be mostly active in heart, kidney, and brain. However, no oxidation of L-2-hydroxyglutarate by rat liver mitochondria was later found by Lindahl and coworkers (7). Furthermore, rat ti...

L-2-hydroxyglutaric aciduria is a metabolic disorder in which L-2-hydroxyglutarate accumulates as a result of a deficiency in FAD-linked L-2-hydroxyglutarate dehydrogenase, a mitochondrial enzyme converting L-2-hydroxyglutarate to alpha-ketoglutarate. The origin of the L-2-hydroxyglutarate, which accumulates in this disorder, is presently unknown. The oxidation-reduction potential of the 2-hydroxyglutarate/alpha-ketoglutarate couple is such that L-2-hydroxyglutarate could potentially be produced through the reduction of alpha-ketoglutarate by a NAD- or NADP-linked oxidoreductase. In fractions of rat liver cytosolic extracts that had been chromatographed on an anion exchanger we detected an enzyme reducing alpha-ketoglutarate in the presence of NADH. This enzyme co-purified with cytosolic L-malate dehydrogenase (cMDH) upon further chromatography on Blue Sepharose. Mitochondrial fractions also contained an NADH-linked, 'alpha-ketoglutarate reductase', which similarly co-purified with mitochondrial L-malate dehydrogenase (mMDH). Purified mMDH catalysed the reduction of alpha-ketoglutarate to L-2-hydroxyglutarate with a catalytic efficiency that was about 10(7)-fold lower than that observed with oxaloacetate. For the cytosolic enzyme, this ratio amounted to 10(8), indicating that this enzyme is more specific. Both cMDH and mMDH are highly active in tissues and alpha-ketoglutarate is much more abundant than oxaloacetate and more concentrated in mitochondria than in the cytosol. As a result of this, the weak activity of mMDH on alpha-ketoglutarate is sufficient to account for the amount of L-2-hydroxyglutarate that is excreted by patients deficient in FAD-linked L-2-hydroxyglutarate dehydrogenase. The latter enzyme appears, therefore, to be responsible for a 'metabolite repair' phenomenon and to belong to the expanding class of 'house-cleaning' enzymes.