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Astrocytes are the major source of L-serine (L-Ser) in the brain: the glycolytic intermediate D-3-phosphoglycerate is converted into L-Ser through the phosphorylated pathway (PP) made up of three enzymes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP), recently proposed to generate a metabolic assembly named serinosome. In the central nervous system, L-Ser is used for a number of functions, including the synthesis of glycine (Gly) and D-serine (D-Ser), the two key NMDAR co-agonists. Here, we used iPSC-derived human astrocytes as a cellular model to evaluate the impact on cell metabolism of the overexpression of each of the three enzymes of the PP as GFP-tagged proteins. The subcellular cytosolic localization of PP enzymes remains unchanged compared to endogenous proteins, while the complex formation is increased in all cases. Notably, among the factors involved, the overexpression of PHGDH appears to play a pivotal role in promoting the serinosome assembly and/or stabilization, highlighting the critical importance of this multi-domain protein. Particularly, the overexpression of each enzyme of the PP alters the cellular metabolism in a specific way. The L-Ser and Gly levels increase more in PHGDH overexpressing cells, in agreement with the known kinetics of the PP. A consistent increase in the TCA cycle, as well as in mitochondrial activities, serine-glycine-one carbon pathway, asparagine, arginine, purine and pyrimidines metabolism is also observed. Peculiar alterations are observed when each enzyme of the PP is overexpressed, strongly supporting the use of human iPSC-derived astrocytes overexpressing the PP pathway enzymes as a valuable cellular model for understanding how Ser glial metabolism occurs in a non-tumor system under both physiological and pathological conditions.

Summary In plants, L‐serine (Ser) biosynthesis occurs through various pathways and is highly dependent on the atmospheric CO2 concentration, especially in C3 species, due to the association of the Glycolate Pathway of Ser Biosynthesis (GPSB) with photorespiration. Characterization of a second plant Ser pathway, the Phosphorylated Pathway of Ser Biosynthesis (PPSB), revealed that it is at the crossroads of carbon, nitrogen, and sulphur metabolism. The PPSB comprises three sequential reactions catalysed by 3‐phosphoglycerate dehydrogenase (PGDH), 3‐phosphoSer aminotransferase (PSAT) and 3‐phosphoSer phosphatase (PSP). PPSB was overexpressed in plants exhibiting two different modes of photosynthesis: Arabidopsis (C3 metabolism), and maize (C4 metabolism), under ambient (aCO2) and elevated (eCO2) CO2 growth conditions. Overexpression in Arabidopsis of the PGDH1 gene alone or PGDH1, PSAT1 and PSP1 in combination increased the Ser levels but also the essential amino acids threonine (aCO2), isoleucine, leucine, lysine, phenylalanine, threonine and methionine (eCO2) compared to the wild‐type. These increases translated into higher protein levels. Likewise, starch levels were also increased in the PPSB‐overexpressing lines. In maize, PPSB‐deficient lines were obtained by targeting PSP1 using Cas9 endonuclease. We concluded that the expression of PPSB in maize male gametophyte is required for viable pollen development. Maize lines overexpressing the AtPGDH1 gene only displayed higher protein levels but not starch at both aCO2 and eCO2 conditions, this translated into a significant rise in the nitrogen/carbon ratio. These results suggest that metabolic engineering of PPSB in crops could enhance nitrogen content, particularly under upcoming eCO2 conditions where the activity of GPSB is limited.

The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the “serinosome”. Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH , the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients’ fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

[This corrects the article DOI: 10.3389/fpls.2018.01830.].

The plant mitochondrial electron transport chain influences carbon and nitrogen metabolism under near anoxic conditions through its involvement in the phytoglobin-nitric oxide cycle, where the respiratory chain reduces nitrite to nitric oxide (NO), followed by NO conversion to nitrate by class 1 phytoglobin. Wild type (WT) and transgenic tobacco ( Nicotiana tabacum L.) with differing amounts of alternative oxidase (AOX) were used to manipulate NO generation under hypoxia, and to examine whether this in turn influenced the gene expression of two stress-related amino acid biosynthetic pathways, the plastid-localized phosphorylated pathway of serine biosynthesis (PPSB), and the γ-aminobutyric acid (GABA) shunt. Under hypoxia, leaf NO emission rate was highest in AOX overexpressors and lowest in AOX knockdowns, with WT showing an intermediate rate. In turn, the rate of NO emission correlated with the degree to which amino acids accumulated. This amino acid accumulation was associated with the increased expression of the enzymes of the stress-related amino acid biosynthetic pathways. However, induction of the PPSB occurred much earlier than the GABA shunt. This work shows that high rates of NO turnover associate with rapid gene induction of the PPSB, establishing a clear link between this pathway and the maintenance of carbon, nitrogen and energy metabolism under hypoxia.

In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5′-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased Km for one of the substrates with invariant kcats for S43R PSAT, and a combination of increased Km and decreased kcat for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.

Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate - serine pathway) is initiated in the cytosol and involves glycerate formation from 3-phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis—the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman’s reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP.

L-serine is an important molecule in all living organisms, and thus its biosynthesis is considered to be regulated according to demand. 3-Phosphoglycerate dehydrogenase (PGDH), the first committed enzyme of the phosphorylated pathway of L-serine biosynthesis, is regulated by negative feedback from L-serine in bacteria. In the case of the vascular plant , two PGDH isozymes out of three are inhibited by L-serine and activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine, suggesting a more complicated regulatory mechanism of L-serine biosynthesis in than in bacteria. However, it remains to be clarified whether the activation mechanism of PGDH by amino acids is conserved in land plants. In this study, we identified the sole isozyme of PGDH in the liverwort (MpPGDH) and elucidated its biochemical characteristics. Mp cDNA encodes a 65.6 kDa protein that contains a putative transit peptide for chloroplast localization. MpPGDH shares 75-80% identity with isozymes and forms a homotetramer . Recombinant MpPGDH exhibited an optimal pH of 9.0, apparent Michaelis constants of 0.49 ± 0.04 and 0.096 ± 0.010 mM for 3-PGA and NAD , respectively, and apparent maximum velocity of 5.65 ± 0.10 μmol⋅min ⋅mg , similar to those of isozymes. Phosphate ions were found to stabilize MpPGDH, suggesting that phosphate ions are also a crucial factor in the regulation of serine biosynthesis via the phosphorylated pathway in . MpPGDH was inhibited by L-serine in a cooperative manner and was activated by L-alanine, L-valine, L-methionine, L-homoserine, and L-homocysteine to a lesser extent than it is in . The results suggest that an ancestral PGDH of land plants was inhibited byL-serine and slightly activated by five other amino acids.