Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

Clinical and biochemical distinctions for a metabolite repair disorder caused by NAXD or NAXE deficiency.

The central cofactors NAD(P)H are prone to damage by hydration, resulting in formation of redox-inactive derivatives designated NAD(P)HX. The highly conserved enzymes NAD(P)HX dehydratase (NAXD) and NAD(P)HX epimerase (NAXE) function to repair intracellular NAD(P)HX. Recently, pathogenic variants in both the NAXD and NAXE genes were associated with rapid deterioration and death after an otherwise trivial fever, infection, or illness in young patients. As more patients are identified, distinct clinical features are emerging depending on the location of the pathogenic variant. In this review, we carefully catalogued the clinical features of all published NAXD deficiency patients and found distinct patterns in clinical presentations depending on which subcellular compartment is affected by the enzymatic deficiency. Exon 1 of NAXD contains a mitochondrial propeptide, and a unique cytosolic isoform is initiated from an alternative start codon in exon 2. NAXD deficiency patients with variants that affect both the cytosolic and mitochondrial isoforms present with neurological defects, seizures and skin lesions. Interestingly, patients with NAXD variants exclusively affecting the mitochondrial isoform present with myopathy, moderate neuropathy and a cardiac presentation, without the characteristic skin lesions, seizures or neurological degeneration. This suggests that cytosolic NAD(P)HX repair may protect from neurological damage, whereas muscle fibres may be more sensitive to mitochondrial NAD(P)HX damage. A deeper understanding of the clinical phenotype may facilitate rapid identification of new cases and allow earlier therapeutic intervention. Niacin-based therapies are promising, but advances in disease modelling for both NAXD and NAXE deficiency may identify more specific compounds as targeted treatments. In this review, we found distinct patterns in the clinical presentations of NAXD deficiency patients based on the location of the pathogenic variant, which determines the subcellular compartment that is affected by the enzymatic deficiency.

Approaches for completing metabolic networks through metabolite damage and repair discovery.

Metabolites are prone to damage, either via enzymatic side reactions, which collectively form the underground metabolism, or via spontaneous chemical reactions. The resulting non-canonical metabolites that can be toxic, are mended by dedicated "metabolite repair enzymes." Deficiencies in the latter can cause severe disease in humans, whereas inclusion of repair enzymes in metabolically engineered systems can improve the production yield of value-added chemicals. The metabolite damage and repair loops are typically not yet included in metabolic reconstructions and it is likely that many remain to be discovered. Here, we review strategies and associated challenges for unveiling non-canonical metabolites and metabolite repair enzymes, including systematic approaches based on high-resolution mass spectrometry, metabolome-wide side-activity prediction, as well as high-throughput substrate and phenotypic screens.

Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program.

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells use coordinated gene expression programs and manage biomolecule supply in order to match the demands of metabolism and translation. Global growth programs complement increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis. How these resources are collectively managed is a fundamental question. The role of the Gcn4/ATF4 transcription factor has been best studied in contexts where cells encounter amino acid starvation. However, high Gcn4 activity has been observed in contexts of rapid cell proliferation, and the roles of Gcn4 in such growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum that maintains metabolic supply in order to sustain translation outputs. By integrating matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lysine and arginine enriched transcripts, which include genes encoding the translation machinery. The Gcn4 dependent lysine and arginine supply thereby maintains the synthesis of the translation machinery. This is required to maintain translation capacity. Gcn4 consequently enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controlled using the same Gcn4 transcriptional outputs that function in distinct contexts.

Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program.

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

Methionine at the Heart of Anabolism and Signaling: Perspectives From Budding Yeast.

Studies using a fungal model, Saccharomyces cerevisiae, have been instrumental in advancing our understanding of sulfur metabolism in eukaryotes. Sulfur metabolites, particularly methionine and its derivatives, induce anabolic programs in yeast, and drive various processes integral to metabolism (one-carbon metabolism, nucleotide synthesis, and redox balance). Thereby, methionine also connects these processes with autophagy and epigenetic regulation. The direct involvement of methionine-derived metabolites in diverse chemistries such as transsulfuration and methylation reactions comes from the elegant positioning and safe handling of sulfur through these molecules. In this mini-review, we highlight studies from yeast that reveal how this amino acid holds a unique position in both metabolism and cell signaling, and illustrate cell fate decisions that methionine governs. We further discuss the interconnections between sulfur and NADPH metabolism, and highlight critical nodes around methionine metabolism that are promising for antifungal drug development.

A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis.

Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that S. cerevisiae cells regulate carbon and nitrogen metabolic homeostasis through tRNA U34-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in a starvation-like metabolic signature. Thiolation mutants have only minor translation defects. However, in these cells phosphate homeostasis genes are strongly down-regulated, resulting in an effectively phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, balance carbon and amino acid metabolic flux and grow optimally, by controlling phosphate availability. These results further biochemically explain how phosphate availability determines a switch to a 'starvation-state'.

2-Oxoglutarate cooperativity and biphasic ammonium saturation of Aspergillus niger NADP-glutamate dehydrogenase are structurally coupled.

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized. The C-terminal sequence (residues 315-460, named the D-segment) was implicated in the AnGDH cooperativity. The D-segment residues largely contribute to the monomer-monomer interface of each trimer in the native hexamer and are far removed from the enzyme active site. The D-segment appears to be a part of the allosteric network responsible for 2-oxoglutarate homotropic interactions in AnGDH. AnGDH and its C415S mutant, but not AtGDH, also showed atypical, biphasic ammonium saturation, particularly at sub-saturating 2-oxoglutarate concentrations. We found that the sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response are tightly coupled; the analysis of AnGDH-AtGDH chimeras ascribes the two features to the AnGDH D-segment. The two non-Michaelis-Menten substrate saturations of AnGDH were influenced by ionic strength. Increase in ionic strength reduced the nH of 2-oxoglutarate saturation as well as abolished the biphasic response, suggesting that polar/ionic interactions determine the allosteric, inter-subunit communications. The biochemical analysis in the context of available structural data implicates the D-segment of AnGDH in the allosteric feature of this enzyme. The coupling of sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response could possibly confer growth advantage to A. niger experiencing carbon and/or nitrogen limitation.

Methionine coordinates a hierarchically organized anabolic program enabling proliferation.

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here we construct the organization of this methionine-mediated anabolic program using yeast. Combining comparative transcriptome analysis and biochemical and metabolic flux-based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH)-dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP)-dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine-mediated anabolic program to be sustained, cells co-opt a "starvation stress response" regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase from Aspergillus niger.

Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study, Aspergillus niger NADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Šresolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space group R32, with unit-cell parameters a=b=173.8, c=241.5 Å, α=ß=90, γ=120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress.

Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.