The human phosphorylated pathway: a multienzyme metabolic assembly for l‐serine biosynthesis

De novo l-serine biosynthesis in the mammalian astrocytes proceeds via a linear, three-step pathway (the phosphorylated pathway) catalysed by 3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase (PSP). The first reaction, catalysed by PHGDH and using the glycolytic intermediate 3-phosphoglycerate, is strongly shifted towards the reagents, and coupling to the following step by PSAT is required to push the equilibrium towards l-serine formation; the last step, catalysed by PSP, is virtually irreversible and inhibited by the final product l-serine. Very little is known about the regulation of the human phosphorylated pathway and the ability of the three enzymes to organise in a complex with potential regulatory functions. Here, the complex formation was investigated in differentiated human astrocytes, by proximity ligation assay, and in vitro on the human recombinant enzymes. The results indicate that the three enzymes co-localise in cytoplasmic clusters that more stably engage PSAT and PSP. Although in vitro analyses based on native PAGE, size exclusion chromatography and cross-linking experiments do not show the formation of a stable complex, kinetic studies of the reconstituted pathway using physiological enzyme and substrate concentrations support cluster formation and indicate that PHGDH catalyses the rate-limiting step while PSP reaction is the driving force for the whole pathway. The enzyme agglomerate assembly of the phosphorylated pathway (the putative 'serinosome') delivers a relevant level of sophistication to the control of l-serine biosynthesis in human cells, a process strictly related to the modulation of the brain levels of d-serine and glycine, the main co-agonists of N-methyl-d-aspartate receptors and various pathological states

Biochemical and cellular studies of three human 3‐phosphoglycerate dehydrogenase variants responsible for pathological reduced L‐serine levels

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches

The Energy Landscape of Human Serine Racemase

Human serine racemase is a pyridoxal 5′-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg2+, Ca2+, anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site

Human serine racemase: regulation by divalent metals, NADH and S-nitrosylation

Human serine racemase (hSR) is a dimeric pyridoxal 5’ phosphate-dependent enzyme involved in the synthesis and metabolism of D-serine, the obligatory co-agonist of the NMDA receptors for glutamate. My PhD work focused on the characterization of some of its allosteric effectors. Divalent cations - Mg2+ and Ca2+ - and ATP both increase enzyme activity. By using analytical gel filtration chromatography, we also found that they shift the quaternary equilibrium towards a tetramer. NADH was also characterized and was found to act as a partial inhibitor, with the 1,4 dihydronicotinamidic ring as structural determinant. The NADH binding site was identified by molecular docking at the dimer interface, close to the ATP binding site. N-substituted 1,4 dihydronicotinamidic derivatives 1-methyl-1,4-dihydronicotinamide and -1,4-dihydronicotinamide mononucleotide were also found to be active, opening the way for the design of allosteric inhibitors. hSR was discovered to be S-nitrosylated at three cysteine residues (Cys113, Cys128 and Cys269), resulting in a biphasic time-dependent inhibition, with the fast phase associated to the nitrosylation of Cys113, as confirmed by the characterization of the C113S mutant. The affinity of nitrosylated hSR for ATP and active site ligands, as glycine or malonate, were found to be comparable to un-modified serine racemase. In the presence of active-site ligands, inhibition by S-nitrosylation was markedly slowed down, suggesting that S-nitrosylation is dependent on the conformational equilibrium. Site-directed mutagenesis of the residues adjacent to Cys113 helped elucidate the structural bases of hSR S-nitrosylation.La serina racemasi umana (hSR) è un enzima dimerico piridossal 5' fosfato coinvolto nella sintesi e nel metabolismo della D-serina, il co-agonista obbligatorio dei recettori NMDA per il glutammato. Il mio lavoro di dottorato si è concentrato sulla caratterizzazione di alcuni dei suoi effettori allosterici. Cationi divalenti - Mg2 + e Ca2 + - e ATP aumentano entrambi l'attività enzimatica. Usando la cromatografia analitica ad esclusione dimensionale, abbiamo osservato che, cationi bivalenti e ATP, spostano l'equilibrio quaternario verso il tetramero. La molecola di NADH, è stata inoltre scoperta agire come inibitore parziale, con l'anello 1,4 diidronicotinamidico come determinante strutturale. Il sito di legame del NADH è stato identificato mediante docking molecolare vicino al sito di legame dell'ATP, all'interfaccia del dimero. Sono stati scoperti essere attivi anche alcuni derivati 1,4 diidronicotinammidici N-sostituiti, quali 1-metil-1,4-diidronicotinammide e β-1,4-diidronicotinammide mononucleotide, aprendo la strada per la progettazione di inibitori allosterici di questo enzima. hSR è stata vista essere S-nitrosilata in tre residui di cisteina (Cys113, Cys128 e Cys269), con conseguente inibizione tempo-dipendente bifasica, la cui fase veloce è associata alla nitrosilazione di Cys113, come confermato dalla caratterizzazione del mutante C113S. L'affinità di hSR nitrosilata per ATP e ligandi del sito attivo, come glicina o malonato, è risultata paragonabile alla serina racemasi non modificata. In presenza di ligandi del sito attivo, l'inibizione della S-nitrosilazione è marcatamente rallentata, suggerendo che la S-nitrosilazione dipenda dall'equilibrio conformazionale. La mutagenesi sito-specifica dei residui adiacenti a Cys113 ha aiutato a chiarire le basi strutturali della nitrosilazione di questo enzima

Human serine racemase is nitrosylated at multiple sites

Serine racemase is a pyridoxal 5'‑phosphate dependent enzyme responsible for the synthesis of d‑serine, a neuromodulator of the NMDA receptors. Its activity is modulated by several ligands, including ATP, divalent cations and protein interactors. The murine orthologue is inhibited by S-nitrosylation at Cys113, a residue adjacent to the ATP binding site. We found that the time course of inhibition of human serine racemase by S-nitrosylation is markedly biphasic, with a fast phase associated with the reaction of Cys113. Unlike the murine enzyme, two additional cysteine residues, Cys269, unique to the human orthologue, and Cys128 were also recognized as S-nitrosylation sites through mass spectrometry and site-directed mutagenesis. The effect of S-nitrosylation on the fluorescence of tryptophan residues and on that of the pyridoxal phosphate cofactor indicated that S-nitrosylation produces a partial interruption of the cross-talk between the ATP binding site and the active site. Overall, it appears that the inhibition results from a conformational change rather than the direct displacement of ATP

Insight of Saffron Proteome by Gel-Electrophoresis

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, -1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds

A Novel Assay for Phosphoserine Phosphatase Exploiting Serine Acetyltransferase as the Coupling Enzyme

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

Progetto Cli_CCHE Quartiere Sant'Antonio L'Italia che verrà: libera interpretazione dell'Italia dell'Antropocene in poster e video mapping

Esposizione dei lavori del Workshop Locale CliCCHE. Progetto Erasmus+ Esposizione e Video Mapping "L'Italia che Verrà", libera interpretazione dell'Italia dell'Antropocene in poster e video mappin

Human serine racemase is allosterically modulated by NADH and reduced nicotinamide derivatives

Serine racemase catalyzes both the synthesis and the degradation of d-serine, an obligatory co-agonist of the glutamatergic NMDA receptors. It is allosterically controlled by adenosine triphosphate (ATP), which increases its activity around 7-fold through a co-operative binding mechanism. Serine racemase has been proposed as a drug target for the treatment of several neuropathologies but, so far, the search has been directed only toward the active site, with the identification of a few, low-affinity inhibitors. Following the recent observation that nicotinamide adenine dinucleotide (reduced form) (NADH) inhibits serine racemase, here we show that the inhibition is partial, with an IC50 of 246 ± 63 μM, several-fold higher than NADH intracellular concentrations. At saturating concentrations of NADH, ATP binds with a 2-fold lower affinity and without co-operativity, suggesting ligand competition. NADH also reduces the weak activity of human serine racemase in the absence of ATP, indicating an additional ATP-independent inhibition mechanism. By dissecting the NADH molecule, we discovered that the inhibitory determinant is the N-substituted 1,4-dihydronicotinamide ring. Particularly, the NADH precursor 1,4-dihydronicotinamide mononucleotide exhibited a partial mixed-type inhibition, with a KI of 18 ± 7 μM. Docking simulations suggested that all 1,4-dihydronicotinamide derivatives bind at the interdimeric interface, with the ring positioned in an unoccupied site next to the ATP-binding site. This newly recognized allosteric site might be exploited for the design of high-affinity serine racemase effectors to finely modulate d-serine homeostasis