Metabolite proofreading, a neglected aspect of intermediary metabolism

Schaftingen, Emile Van; Rim, Rzem; Marbaix, Alexandre; Collard, François; Veiga‐da‐Cunha, Maria; Linster, Carole L.

doi:10.1007/s10545-012-9571-1

Cited by 69 publications

(66 citation statements)

References 48 publications

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“…Over the past years, a number of so-called "metabolite repair enzymes" have been identified whose apparently exclusive role is to convert useless and potentially toxic side products formed endogenously by metabolic enzymes (65,74). These side products are formed because metabolic enzymes are not perfectly specific, and they can act on compounds that resemble their main physiological substrate or they can catalyze more than one type of reaction on the main physiological substrate.…”

Section: Identification Of a New Type Of Enzymatic Activity For Degramentioning

confidence: 99%

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Becker-Kettern

Paczia

Conrotte

et al. 2016

Journal of Biological Chemistry

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122

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The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3⌬ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2⌬ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to ␣-ketoglutarate. Depletion of D-lactate levels in the dld3⌬, but not in the dld2⌬ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to ␣-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce ␣-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1. 2-Hydroxyglutarate (2HG)3 is a 5-carbon dicarboxylic acid that was first detected in human urine in the late 1970s (1).Because of the hydroxyl group on the second carbon, 2HG exists under two enantiomeric configurations (L or D) that can be separated by gas or liquid chromatography after derivatization with another chiral compound and that can therefore be differentially assayed in biological samples using GC-MS or LC-MS methods (2, 3). The interest in 2HG increased when it was found to accumulate in urine of patients with suspected inborn errors of metabolism (4, 5). Most 2-hydroxyglutaric aciduria patients present elevations of either L-2HG or D-2HG in their extracellular fluids, and the clinical phenotype depends on the configuration of the accumulated organic acid. More recently, cases of "combined D,L-hydroxyglutaric aciduria" have been reported (6).2-Hydroxyglutaric acidurias remained enigmatic diseases because neither L-2HG nor D-2HG are intermediates of any known metabolic pathway, and the causal gene deficiencies were only discovered many years after the first patient case reports. It is now established that L-2-hydroxyglutaric aciduria is caused by loss-of-function mutations in the L2HGDH gene, encoding a specific L-2HG dehydrogenase, whereas in many cases D-2-hydroxyglutaric aciduria results from loss-of-function mutations in the D2H...

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Section: Identification Of a New Type Of Enzymatic Activity For Degramentioning

confidence: 99%

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Becker-Kettern

Paczia

Conrotte

et al. 2016

Journal of Biological Chemistry

Self Cite

122

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“…It is becoming increasingly clear that such metabolite damage is countered in all organisms by a suite of damage-control systems (Galperin et al, 2006;Linster et al, 2013;Van Schaftingen et al, 2013). These systems fall into two categories: those that repair metabolite damage and those that preempt it .…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

et al. 2014

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RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

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“…Recent research has unveiled the importance of metabolite repair in intermediary metabolism (12,13). According to this concept, enzymes of intermediary metabolism are not perfectly specific and tend to act on intracellular compounds resembling their true substrate.…”

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confidence: 99%

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

Peracchi

Veiga‐da‐Cunha

Kuhara

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.metabolite repair | deaminated glutathione | amidase | aminotransferases

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Metabolite proofreading, a neglected aspect of intermediary metabolism

Cited by 69 publications

References 48 publications

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

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