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

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

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

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

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95

Human serine racemase (hSR) is a pyridoxal-5′-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg2+, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction

Magnesium and calcium ions differentially affect human serine racemase activity and modulate its quaternary equilibrium toward a tetrameric form

Serine racemase is the pyridoxal 5′-phosphate dependent enzyme that catalyzes both production and catabolism of D-serine, a co-agonist of the NMDA glutamate receptors. Mg2 +, or, alternatively, Ca2 +, activate human serine racemase by binding both at a specific site and – as ATP-metal complexes – at a distinct ATP binding site. We show that Mg2 + and Ca2 + bind at the metal binding site with a 4.5-fold difference in affinity, producing a similar thermal stabilization and partially shifting the dimer-tetramer equilibrium in favour of the latter. The ATP-Ca2 + complex produces a 2-fold lower maximal activation in comparison to the ATP-Mg2 + complex and exhibits a 3-fold higher EC50. The co-presence of ATP and metals further stabilizes the tetramer. In consideration of the cellular concentrations of Mg2 + and Ca2 +, even taking into account the fluctuations of the latter, these results point to Mg2 + as the sole physiologically relevant ligand both at the metal binding site and at the ATP binding site. The stabilization of the tetramer by both metals and ATP-metal complexes suggests a quaternary activation mechanism mediated by 5′-phosphonucleotides similar to that observed in the distantly related prokaryotic threonine deaminases. This allosteric mechanism has never been observed before in mammalian fold type II pyridoxal 5′-phosphate dependent enzymes