Identification of the enzyme and the gene defect in L-2-hydroxyglutaric aciduria
(2006)
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- L-2-hydroxyglutaric aciduria is a neurometabolic disorder characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma and cerebrospinal fluid. The enzymatic defect leading to this condition is unknown, as knowledge of the origin and fate of L-2-hydroxyglutarate is lacking. The aim of this thesis is to identify the biochemical and the genetic defects in L-2-hydroxyglutaric aciduria and to determine the origin of L-2-hydroxyglutarate, which accumulates in this disorder. <br>Chromatography of a frozen rat liver extract on DEAE-Sepharose separated two major peaks of enzymes catalyzing the detritiation of DL-2-hydroxy[2-3H]glutarate. The first peak represented the enzyme present in the flow-through that acted on the L-isomer, the second peak eluted at high salt concentrations corresponded to D-2-hydroxyglutarate dehydrogenase. The first enzyme catalyzed specifically the oxidation of L-2-hydroxyglutarate to ?-ketoglutarate using FAD as a cofactor. It was mainly expressed in liver and kidney, but also at lower levels in heart, brain and some other tissues. Subcellular fractionation indicated that the liver enzyme was present in mitochondria where it is bound to its membrane. Based on this information, a database search led us to the identification of a gene encoding a human hypothetical protein homologous to bacterial FAD-dependent malate dehydrogenases and targeted to the mitochondria. Initially, we demonstrated that the gene encoding this protein, present on chromosome 14q22.1, is located in a region homozygous in patients with L-2-hydroxyglutaric aciduria from two consanguineous families. In addition, we identified three homozygous mutations that replace a highly conserved residue (Lys81Glu; Glu176Asp) or removed exon 9 in patients affected with L-2-hydroxyglutaric aciduria from three distinct families. <br>We transfected embryonic kidney cells with the cDNA encoding the product of the human gene. The overexpression experiments led to greater than a 15-fold increase in L-2-hydroxyglutarate dehydrogenase activity. The overexpressed enzyme had similar biochemical characteristics to the rat liver enzyme, including sensitivity to FAD and association with membranes. Transfection of enzyme forms containing mutations found in patients with L-2-hydroxyglutaric aciduria did not cause any increase in the L-2-hydroxyglutarate dehydrogenase activity. These results provide evidence for the fact that L-2-hydroxyglutarate is normally metabolized to ?-ketoglutarate in mammalian tissues and that L-2-hydroxyglutaric aciduria is due to L-2-hydroxyglutarate dehydrogenase deficiency.<br>To identify the origin of L-2-hydroxyglutarate, we searched for an enzyme able to reduce ?-ketoglutarate. Such an enzymatic activity was indeed found in liver extracts and shown to correspond to a side activity of L-malate dehydrogenase, most particularly the mitochondrial form of this enzyme. Thus, our findings suggest that the presence of L-2-hydroxyglutarate results from a side-reaction whereby L-malate dehydrogenase is capable of reducing ?-ketoglutarate and that L-2-hydroxyglutarate dehydrogenase catalyzes a reaction of metabolite repair. L-2-hydroxyglutaric aciduria is therefore a disorder
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Rzem, R. (2006). Identification of the enzyme and the gene defect in L-2-hydroxyglutaric aciduria. https://hdl.handle.net/2078.5/112154