Identification of the enzyme and the gene defect in L-2-hydroxyglutaric aciduria

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. 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. 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.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 disorderDoctorat en sciences biomédicales (SBIM 3)--UCL, 200

The gene mutated in l-2-hydroxyglutaric aciduria encodes l-2-hydroxyglutarate dehydrogenase.

The biochemical defect in L-2-hydroxyglutaric aciduria is still unknown, but the mutated gene has recently been identified on chromosome 14q22. Transfection of human embryonic kidney (HEK) cells with a cDNA encoding the product of the human gene led to a>15-fold increase in L-2-hydroxyglutarate dehydrogenase activity. The overexpressed enzyme had similar biochemical characteristics (including sensitivity to FAD and association with membranes) as the rat liver enzyme. Western blot analysis indicated that it is processed through the removal of a N-terminal approximately 4 kDa fragment, in agreement with a mitochondrial localization. Transfection experiments indicated that the mutations (K81E, E176D, Delta-exon9) found in patients with L-2-hydroxyglutaric aciduria suppressed L-2-hydroxyglutarate dehydrogenase activity. Western blot analysis showed that the three mutated proteins were expressed to various degrees in HEK cells, but were abnormally processed. Taken together, these data indicate that L-2-hydroxyglutaric aciduria is due to a deficiency in L-2-hydroxyglutarate dehydrogenase

HDHD1, which is often deleted in X-linked ichthyosis, encodes a pseudouridine-5 '-phosphatase

Pseudouridine. the fifth-most abundant nucleoside in RNA, is not metabolized in mammals. but is excreted intact in urine The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5'-phosphate, a potential intermediate in RNA degradation We show that human erythrocytes contain a pseudouridine-5'-phosphatase displaying a K-m <= mu M for its substrate The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate. suggesting that it belonged to the 'haloacid dehalogenase' family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant I-I D I dephosphorylated pseudouridine 5'-phosphate with a k(cat) of 1 6 s(-1). a K-m 0.3 mu M and a catalytic efficiency at least 1000-fold higher than that or which it acted on other phosphate esters, including 5'-UMP. The molecular identity of pseudouridine-5'-phosphatase was confirmed by the finding that its activity was negligible (<10% of controls) in extracts of B-cell lymphoblasts or erythrocytes both X-linked ichthyosis patients harbouring a combined deletion of the STS gene (the X-linked ichthyosis gene) and the HDHD1 Furthermore, pseudouridine-5'-phosphatase activity was 1.5-fold higher in erythrocytes from women compared with men, in agreement with the HDHD1 gene undergoing only partial inactivation in females In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RN

L-2-Hydroxyglutaric aciduria, a disorder of metabolite repair.

The neurometabolic disorder L: -2-hydroxyglutaric aciduria is caused by mutations in a gene present on chromosome 14q22.1 and encoding L: -2-hydroxyglutarate dehydrogenase. This FAD-linked mitochondrial enzyme catalyses the irreversible conversion of L: -2-hydroxyglutarate to alpha-ketoglutarate. The formation of L: -2-hydroxyglutarate results from a side-activity of mitochondrial L: -malate dehydrogenase, the enzyme that interconverts oxaloacetate and L: -malate, but which also catalyses, very slowly, the NADH-dependent conversion of alpha-ketoglutarate to L: -2-hydroxyglutarate. L: -2-Hydroxyglutarate has no known physiological function in eukaryotes and most prokaryotes. Its accumulation is toxic to the mammalian brain, causing a leukoencephalopathy and increasing the susceptibility to develop tumours. L: -2-Hydroxyglutaric aciduria appears to be the first disease of 'metabolite repair'

L-2-hydroxyglutaric aciduria, a defect of metabolite repair.

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

Many fructosamine 3-kinase homologues in bacteria are ribulosamine/erythrulosamine 3-kinases potentially involved in protein deglycation

The purpose of this work was to identify the function of bacterial homologues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate

A mouse model of L-2-hydroxyglutaric aciduria, a disorder of metabolite repair

The purpose of the present work was to progress in our understanding of the pathophysiology of L-2-hydroxyglutaric aciduria, due to a defect in L-2-hydroxyglutarate dehydrogenase, by creating and studying a mouse model of this disease. L-2-hydroxyglutarate dehydrogenase-deficient mice (l2hgdh-/-) accumulated L-2-hydroxyglutarate in tissues, most particularly in brain and testis, where the concentration reached ≈ 3.5 μmol/g. Male mice showed a 30% higher excretion of L-2-hydroxyglutarate compared to female mice, supporting that this dicarboxylic acid is partially made in males by lactate dehydrogenase C, a poorly specific form of this enzyme exclusively expressed in testes. Involvement of mitochondrial malate dehydrogenase in the formation of L-2-hydroxyglutarate was supported by the commensurate decrease in the formation of this dicarboxylic acid when down-regulating this enzyme in mouse l2hgdh-/- embryonic fibroblasts. The concentration of lysine and arginine was markedly increased in the brain of l2hgdh-/- adult mice. Saccharopine was depleted and glutamine was decreased by ≈ 40%. Lysine-α-ketoglutarate reductase, which converts lysine to saccharopine, was inhibited by L-2-hydroxyglutarate with a Ki of ≈ 0.8 mM. As low but significant activities of the bifunctional enzyme lysine-α-ketoglutarate reductase/saccharopine dehydrogenase were found in brain, these findings suggest that the classical lysine degradation pathway also operates in brain and is inhibited by the high concentrations of L-2-hydroxyglutarate found in l2hgdh-/- mice. Pathological analysis of the brain showed significant spongiosis. The vacuolar lesions mostly affected oligodendrocytes and myelin sheats, as in other dicarboxylic acidurias, suggesting that the pathophysiology of this model of leukodystrophy may involve irreversible pumping of a dicarboxylate in oligodendrocytes. Neurobehavioral testing indicated that the mice mostly suffered from a deficit in learning capacity. In conclusion, the findings support the concept that L-2-hydroxyglutaric aciduria is a disorder of metabolite repair. The accumulation of L-2-hydroxyglutarate exerts toxic effects through various means including enzyme inhibition and glial cell swelling

Formation and breakdown of L-2-hydroxyglutarate and interference of this compound with lysine metabolism.

<p>The scheme shows how L-2-hydroxyglutarate is formed and degraded. It also shows the initial steps of the major lysine catabolic pathway (via saccharopine) present in mammalian tissues and of the minor pathway (via L-pipecolate) present in brain and the inhibition exerted by L-2-hydroxyglutarate. 1. Lysine-α-ketoglutarate reductase; 2. Saccharopine dehydrogenase; 3. α-Aminoadipate semialdehyde dehydrogenase; 4. α-Aminoadipate transaminase; 5. Imine reductase; 6. L-Pipecolate oxidase.</p

Amino acids in brain, testis and plasma of <i>l2hgdh</i>^<i>+/+</i> and <i>l2hgdh</i>^{<i>−/−</i>} mice.

<p>Same experimental procedure as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119540#pone.0119540.g005" target="_blank">Fig. 5</a>.</p><p>Values are means ± SEM for the number of samples indicated that are shown in nmol/g (brain, testis) or nmol/ml (plasma).</p><p>*Significantly different values (alpha < 0.0016) are indicated with an asterisk.</p><p>The analysis was performed with the Student t-test with a Bonferroni correction for multiple testing (32 parameters).</p><p>Amino acids in brain, testis and plasma of <i>l2hgdh</i>^<i>+/+</i> and <i>l2hgdh</i>^{<i>−/−</i>} mice.</p

Presence of the bifunctional enzyme lysine-α-ketoglutarate reductase/saccharopine dehydrogenase in brain (A, B) and inhibition of lysine-α-ketoglutarate reductase by L-2-hydroxyglutarate (C).

<p>(A) and (B) show the elution profile of lysine-α-ketoglutarate reductase (LαKGR) and saccharopine dehydrogenase (SD) from Blue Trisacryl columns on which a mouse liver (A) or a mouse brain (B) extract have been applied. (C) Partially purified lysine-α-ketoglutarate reductase from mouse liver was assayed at 30°C in the presence of 25 mM Hepes, pH 8.0, 0.15 mM NADPH, 1 mM dithiothreitol, 10 mM lysine (Lys) and 2, 5 or 10 mM α-ketoglutarate (α-KG) as indicated and increasing concentrations of L-2-hydroxyglutarate. The effect of L-2-hydroxyglutarate was also tested in the presence of 25 mM lysine and 2 mM α-ketoglutarate and that of D-2-hydroxyglutarate with 10 mM lysine and 2 mM α-ketoglutarate.</p