A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD+-dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease.

Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite.

Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

Metabolite Repair Enzymes Control Metabolic Damage in Glycolysis.

Hundreds of metabolic enzymes work together smoothly in a cell. These enzymes are highly specific. Nevertheless, under physiological conditions, many perform side-reactions at low rates, producing potentially toxic side-products. An increasing number of metabolite repair enzymes are being discovered that serve to eliminate these noncanonical metabolites. Some of these enzymes are extraordinarily conserved, and their deficiency can lead to diseases in humans or embryonic lethality in mice, indicating their central role in cellular metabolism. We discuss how metabolite repair enzymes eliminate glycolytic side-products and prevent negative interference within and beyond this core metabolic pathway. Extrapolating from the number of metabolite repair enzymes involved in glycolysis, hundreds more likely remain to be discovered that protect a wide range of metabolic pathways.

Impaired glucose-1,6-biphosphate production due to bi-allelic PGM2L1 mutations is associated with a neurodevelopmental disorder.

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.

SGLT5 is the renal transporter for 1,5-anhydroglucitol, a major player in two rare forms of neutropenia.

Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) and severe congenital neutropenia type 4 (SCN4), associated with deficiencies of the glucose-6-phosphate transporter (G6PT/SLC37A4) and the phosphatase G6PC3, respectively, are the result of the accumulation of 1,5-anhydroglucitol-6-phosphate in neutrophils. This is an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol in blood. 1,5-AG is presumed to be reabsorbed in the kidney by a sodium-dependent-transporter of uncertain identity, possibly SGLT4/SLC5A9 or SGLT5/SLC5A10. Lowering blood 1,5-AG with an SGLT2-inhibitor greatly improved neutrophil counts and function in G6PC3-deficient and GSD1b patients. Yet, this effect is most likely mediated indirectly, through the inhibition of the renal 1,5-AG transporter by glucose, when its concentration rises in the renal tubule following inhibition of SGLT2. To identify the 1,5-AG transporter, both human and mouse SGLT4 and SGLT5 were expressed in HEK293T cells and transport measurements were performed with radiolabelled compounds. We found that SGLT5 is a better carrier for 1,5-AG than for mannose, while the opposite is true for human SGLT4. Heterozygous variants in SGLT5, associated with a low level of blood 1,5-AG in humans cause a 50-100% reduction in 1,5-AG transport activity tested in model cell lines, indicating that SGLT5 is the predominant kidney 1,5-AG transporter. These and other findings led to the conclusion that (1) SGLT5 is the main renal transporter of 1,5-AG; (2) frequent heterozygous mutations (allelic frequency > 1%) in SGLT5 lower blood 1,5-AG, favourably influencing neutropenia in G6PC3 or G6PT deficiency; (3) the effect of SGLT2-inhibitors on blood 1,5-AG level is largely indirect; (4) specific SGLT5-inhibitors would be more efficient to treat these neutropenias than SGLT2-inhibitors.

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates.

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

Convergent evolution of zoonotic Brucella species toward the selective use of the pentose phosphate pathway.

Mechanistic understanding of the factors that govern host tropism remains incompletely understood for most pathogens. Brucella species, which are capable of infecting a wide range of hosts, offer a useful avenue to address this question. We hypothesized that metabolic fine-tuning to intrahost niches is likely an underappreciated axis underlying pathogens' ability to infect new hosts and tropism. In this work, we compared the central metabolism of seven Brucella species by stable isotopic labeling and genetics. We identified two functionally distinct groups, one overlapping with the classical zoonotic species of domestic livestock that exclusively use the pentose phosphate pathway (PPP) for hexose catabolism, whereas species from the second group use mostly the Entner-Doudoroff pathway (EDP). We demonstrated that the metabolic dichotomy among Brucellae emerged after the acquisition of two independent EDP-inactivating mutations in all classical zoonotic species. We then examined the pathogenicity of key metabolic mutants in mice and confirmed that this trait is tied to virulence. Altogether, our data are consistent with the hypothesis that the PPP has been incrementally selected over the EDP in parallel to Brucella adaptation to domestic livestock.

ECHDC1 knockout mice accumulate ethyl-branched lipids and excrete abnormal intermediates of branched-chain fatty acid metabolism.

The cytosolic enzyme ethylmalonyl-CoA decarboxylase (ECHDC1) decarboxylates ethyl- or methyl-malonyl-CoA, two side products of acetyl-CoA carboxylase. These CoA derivatives can be used to synthesize a subset of branched-chain fatty acids (FAs). We previously found that ECHDC1 limits the synthesis of these abnormal FAs in cell lines, but its effects in vivo are unknown. To further evaluate the effects of ECHDC1 deficiency, we generated knockout mice. These mice were viable, fertile, showed normal postnatal growth, and lacked obvious macroscopic and histologic changes. Surprisingly, tissues from wild-type mice already contained methyl-branched FAs due to methylmalonyl-CoA incorporation, but these FAs were only increased in the intraorbital glands of ECHDC1 knockout mice. In contrast, ECHDC1 knockout mice accumulated 16-20-carbon FAs carrying ethyl-branches in all tissues, which were undetectable in wild-type mice. Ethyl-branched FAs were incorporated into different lipids, including acylcarnitines, phosphatidylcholines, plasmanylcholines, and triglycerides. Interestingly, we found a variety of unusual glycine-conjugates in the urine of knockout mice, which included adducts of ethyl-branched compounds in different stages of oxidation. This suggests that the excretion of potentially toxic intermediates of branched-chain FA metabolism might prevent a more dramatic phenotype in these mice. Curiously, ECHDC1 knockout mice also accumulated 2,2-dimethylmalonyl-CoA. This indicates that the broad specificity of ECHDC1 might help eliminate a variety of potentially dangerous branched-chain dicarboxylyl-CoAs. We conclude that ECHDC1 prevents the formation of ethyl-branched FAs and that urinary excretion of glycine-conjugates allows mice to eliminate potentially deleterious intermediates of branched-chain FA metabolism.

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate.

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.

Treating neutropenia and neutrophil dysfunction in glycogen storage disease type Ib with an SGLT2 inhibitor.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

Successful use of empagliflozin to treat neutropenia in two G6PC3-deficient children: Impact of a mutation in SGLT5.

Neutropenia and neutrophil dysfunction found in deficiencies in G6PC3 and in the glucose-6-phosphate transporter (G6PT/SLC37A4) are due to accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol present in blood. Lowering blood 1,5-AG with an SGLT2 inhibitor greatly improved neutrophil counts and function in G6PC3-deficient mice and in patients with G6PT-deficiency. We evaluate this treatment in two G6PC3-deficient children. While neutropenia was severe in one child (PT1), which was dependent on granulocyte cololony-stimulating factor (GCSF), it was significantly milder in the other one (PT2), which had low blood 1,5-AG levels and only required GCSF during severe infections. Treatment with the SGLT2-inhibitor empagliflozin decreased 1,5-AG in blood and 1,5-AG6P in neutrophils and improved (PT1) or normalized (PT2) neutrophil counts, allowing to stop GCSF. On empagliflozin, both children remained infection-free (>1 year - PT2; >2 years - PT1) and no side effects were reported. Remarkably, sequencing of SGLT5, the gene encoding the putative renal transporter for 1,5-AG, disclosed a rare heterozygous missense mutation in PT2, replacing the extremely conserved Arg401 by a histidine. The higher urinary clearance of 1,5-AG explains the more benign neutropenia and the outstanding response to empagliflozin treatment found in this child. Our data shows that SGLT2 inhibitors are an excellent alternative to treat the neutropenia present in G6PC3-deficiency.

Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency.

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-KM hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-KM hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.

Variants in the ethylmalonyl-CoA decarboxylase (ECHDC1) gene: a novel player in ethylmalonic aciduria?

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short-chain acyl-CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl-CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss-of-function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven.

SLC13A3 variants cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation.

OBJECTIVE: SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. METHODS: Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. RESULTS: WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. INTERPRETATION: SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics.

Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.

Inborn errors of metabolite repair.

It is traditionally assumed that enzymes of intermediary metabolism are extremely specific and that this is sufficient to prevent the production of useless and/or toxic side-products. Recent work indicates that this statement is not entirely correct. In reality, enzymes are not strictly specific, they often display weak side activities on intracellular metabolites (substrate promiscuity) that resemble their physiological substrate or slowly catalyse abnormal reactions on their physiological substrate (catalytic promiscuity). They thereby produce non-classical metabolites that are not efficiently metabolised by conventional enzymes. In an increasing number of cases, metabolite repair enzymes are being discovered that serve to eliminate these non-classical metabolites and prevent their accumulation. Metabolite repair enzymes also eliminate non-classical metabolites that are formed through spontaneous (ie, not enzyme-catalysed) reactions. Importantly, genetic deficiencies in several metabolite repair enzymes lead to 'inborn errors of metabolite repair', such as L-2-hydroxyglutaric aciduria, D-2-hydroxyglutaric aciduria, 'ubiquitous glucose-6-phosphatase' (G6PC3) deficiency, the neutropenia present in Glycogen Storage Disease type Ib or defects in the enzymes that repair the hydrated forms of NADH or NADPH. Metabolite repair defects may be difficult to identify as such, because the mutated enzymes are non-classical enzymes that act on non-classical metabolites, which in some cases accumulate only inside the cells, and at rather low, yet toxic, concentrations. It is therefore likely that many additional metabolite repair enzymes remain to be discovered and that many diseases of metabolite repair still await elucidation.

Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).