D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

中文翻译：

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人类D-2-羟基戊二酸脱氢酶和两种与疾病相关的变异的生化特征揭示了D-2-羟基戊二酸尿症的分子原因。

D-2-羟基戊二酸尿症是一种神经代谢疾病，其特征在于D-2-羟基戊二酸（D-2HG）在人线粒体中的积累。在人类中检测到D-2HG的水平升高，这些人类在编码异柠檬酸脱氢酶，柠檬酸盐载体，电子转移黄素蛋白（ETF）及其下游电子受体ETF-泛醌氧化还原酶或D-2-羟基戊二酸脱氢酶（hD2HGDH）的基因中表现出点突变。然而，尽管已经广泛研究了前四种蛋白质中几种氨基酸替代物的致病性，但对某些点突变对hD2HGDH的生化特性的影响知之甚少。因此，我们重组生产了野生型hD2HGDH以及两个最近发现的与疾病相关的变异体（hD2HGDH-I147S和-V444A），并进行了详细的生化鉴定。我们可以证明hD2HGDH是一种FAD依赖性蛋白，它能够催化D-2HG和D-乳酸分别氧化为α-酮戊二酸和丙酮酸。获得了这两种变体作为载脂蛋白，因此具有催化活性。FAD的添加无法恢复变体的酶活性，表明辅因子结合位点被单个氨基酸替代所损害。进一步的分析表明，两种变体均形成聚集体，这些聚集体显然无法结合FAD辅助因子。以来，D-2-羟基戊二酸尿症也可能是由于ETF或其下游电子受体ETF-泛醌氧化还原酶的功能丧失而引起的，ETF可以充当还原型hD2HGDH的同源电子受体。在这里，我们显示hD2HGDH直接还原重组人ETF，从而在D-2-羟基戊二酸的氧化与线粒体电子传输链之间建立了代谢联系。