MINIREVIEW D-Amino acids in the brain: the biochemistry of brain serine racemase Florian Baumgart and Ignacio Rodrı ´ guez-Crespo Departamento de Bioquı ´ mica y Biologı ´ a Molecular, Facultad de Ciencias Quı ´ micas, Universidad Complutense de Madrid, Spain The initial purification of brain mammalian serine racemase was performed by Wolosker, Snyder and coworkers using 60 brains obtained from rats [1]. This seminal work permitted the isolation of a homo- geneous protein preparation that displayed the ability to isomerize l-serine into its enantiomeric d-serine counterpart. In addition, the authors established the molecular mass of the enzyme, its pH and temperature dependence, the presence of bound pyridoxal-5¢ phos- phate (PLP) and the exquisite activity regulation exerted by reagents that react with free SH groups, such as oxidized glutathione. The subsequent mole- cular cloning of mouse brain serine racemase, as well as the comparison with PLP-containing racemases from other organisms, led to the identification of K56 as the lysine residue that formed the Schiff base with the PLP moiety [2]. The first recombinant expression and purification experiment was performed by Wolos- ker and coworkers using HEK293 cells transfected with a serine racemase–glutathione S-transferase plasmid [3]. Keywords AMPA receptor; astrocytes; ATP; calcium activation; D-serine; gliotransmitters; GRIP; NMDA receptor; PDZ interaction; serine racemase Correspondence I. Rodrı ´ guez-Crespo, Departamento de Bioquı ´ mica y Biologı ´ a Molecular, Facultad de Ciencias Quı ´ micas, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain Fax: +34 91 394 4159 Tel: +34 91394 4137 E-mail: nacho@bbm1.ucm.es (Received 30 January 2008, revised 3 April 2008, accepted 4 April 2008) doi:10.1111/j.1742-4658.2008.06517.x It has been recently established that in various brain regions d-serine, the product of serine racemase, occupies the so-called ‘glycine site’ within N-methyl d-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5¢ phosphate-containing enzyme that catalyzes the racemization of l-serine to d-serine. It has also been shown to catalyze the a,b-elimina- tion of water from l-serine or d-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5¢ phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein inter- acting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above. Abbreviations [Ca 2+ ] cyt , cytosolic calcium concentration; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPAR, AMPA receptor; GluR2, glutamate receptor subunit 2; Golga3, Golgin subfamily A member 3; GRIP, glutamate receptor interacting protein; GSNO, S-nitroso- glutathione; NO, nitric oxide; PDZ, PSD95 ⁄ disc large ⁄ ZO-1; PICK1, protein interacting with C kinase 1; PKC, protein kinase C; PLP, pyridoxal-5¢ phosphate. 3538 FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS The purified enzyme was extremely efficient in terms of the elimination reaction, using l-serine-O-sulfate as a substrate and producing pyruvate plus ammonia. However, this purified enzyme failed to catalyze the elimination when l-serine was used as a substrate. A major breakthrough was the observation that both divalent cations and nucleotides were actually cofac- tors of serine racemase [4]. Both the racemase and eliminase reactions of recombinant serine racemase expressed in mammalian cells when l-serine was used as a substrate were activated to similar levels in the presence of divalent cations such as calcium and mag- nesium [4]. This activation by divalent cations was also observed when serine racemase was recombinantly expressed and purified from Escherichia coli [5] or when it was purified from mouse brain [6]. When recombinant serine racemase produced in mammalian cells was used, in terms of d-serine synthesis (racemase activity) both magnesium and ATP independently acti- vated the enzyme and their effect was additive. Even in the presence of the chelating agent EDTA, ATP was still able to increase serine racemase activity [4]. In the absence of added ATP, mammalian cells expressing serine racemase became activated, in terms of pyruvate production, at about 100 lm magnesium. However, in the presence of the nucleotide, the amount of magne- sium needed for half activation was close to 10 lm [4]. Similar data were obtained when the enzyme purified from bacteria was used: in the absence of added ATP, calcium activated the racemase activity of the enzyme at a half-maximal concentration (EC 50 ) of about 26 lm, although using changes in tryptophan fluores- cence a binding constant for calcium to serine race- mase was narrowed down to about 6 lm [5]. The physiological activation of brain serine racemase by divalent cations is described in detail below. Very recently, no fewer than six PLP-containing enzymes having broad sequence homology with human brain serine racemase have been cloned and recombi- nantly expressed. Three recombinant plant serine race- mases have recently been characterized: those of Arabidopsis thaliana, Hordeum vulgare (barley) and Oryza sativa (rice) [7,8]. An aspartate racemase that has a very high homology with brain serine racemase has recently been cloned and characterized from a bivalve mollusk [9]. The so-called serine racemase from Saccha- romyces cerevisiae has been recombinantly expressed in E. coli and characterized, and its properties seem to indicate that it is a paralog rather than an ortholog of mammalian serine racemases [10]. Likewise, the serine racemase from the hyperthermophylum Pyrobacu- lum islandicum was both purified and recombinantly expressed, and the isolated enzymes were characterized [11]. Finally, the coordinates of the 3D structure of ser- ine racemase from Schizosaccharomyces pombe, another enzyme that displays high homology with mammalian serine racemase, have been recently deposited (Protein Data Bank code 1WTC). Sequence comparison allowed us to rationalize the dependence of each of these enzymes on divalent cations and nucleotides, and on their binding to other interacting proteins. Regulation of serine racemase by divalent cations and nucleotides The sequence comparison of human brain serine race- mase with selected homologous proteins is depicted in Fig. 1. We recently used the coordinates obtained from the crystal structure of the Mg 2+ -bound S. pombe serine racemase and the Ca 2+ -bound Thermus thermo- philus threonine deaminase to identify the equivalent positions within mammalian serine racemase that would bind the divalent cation [12]. We were able to predict that the metal is hexavalently coordinated and that the cation-binding site is formed by two carboxyl- ate-containing residues, a main-chain carbonyl oxygen and three well-ordered water molecules. The positions involved in the interaction with the divalent cation are marked in orange in Fig. 1. In human serine racemase, the residues predicted to be directly involved in cal- cium binding are Glu210, Asp216 and Ala214. Consis- tent with this prediction, these three residues, which are conserved in plant and yeast serine racemases, would be responsible for the Ca 2+ ⁄ Mg 2+ racemase activation observed for these enzymes [7,8,10]. Con- versely, the absence of these residues in the bivalve and Pyrobaculum serine racemases is in agreement with the absence of increased racemization of these enzymes induced by Ca 2+ or Mg 2+ [9,11]. The enzyme activation by nucleotides is somehow more puzzling. For instance, the activation of the homologous protein E. coli Thr dehydratase by AMP was observed as early as 1949 [13]. However, in this bacterial enzyme the nucleotide exerted an allosteric role, promoting protein oligomerization and activating the enzyme [14]. In the absence of AMP, the K m of E. coli Thr deaminase for Thr was 70 mm and it decreased to 5 mm in the presence of the nucleotide [15]. The residues participating in the binding of nucle- otides (shown in red in Fig. 1) could also be predicted because the crystal structure of the S. pombe serine racemase has the nucleotide AMPpcp bound [12]. Interestingly, most of the nucleotide-binding sites are conserved in all the enzymes. However, although mam- malian serine racemase is strongly activated by nucleo- tides [4,6,12] this is not the case in their plant F. Baumgart and I. Rodrı ´ guez-Crespo Biochemistry of brain serine racemase FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS 3539 Fig. 1. Sequence alignment of human serine racemase (gi:11345492), Arabidopsis thaliana serine racemase (gi:84458483), Hordeum vulgare (barley) serine racemase (gi:148356707), Bivalve (Scapharca broughtonii) aspartate racemase (gi:86439930), Saccharomyces cerevisiae homolog of serine racemase (gi:151941446), Schizosaccharomyces pombe serine racemase (gi:71041740) and Pyrobaculum islandicum ser- ine racemase (gi:83582728). Based on the crystal structure of S. pombe serine racemase, green arrows depict b-strands and yellow barrels depict a-helices. The modelling has previously been described in detail by Baumgart et al. [12]. The residues involved in calcium binding are shown in orange and those involved in nucleotide binding are shown in red. Residues involved in the binding to the PLP moiety are shown in blue, whereas those involved in protein–protein interaction are shown in green. The first four amino acids of the barley serine racemase and the final 88 amino acids of the Pyrobaculum serine racemase are omitted for clarity. Alignment was performed using the CLUSTAL software. Biochemistry of brain serine racemase F. Baumgart and I. Rodrı ´ guez-Crespo 3540 FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS orthologs [7,8]. In fact, both the bivalve and Pyrobacu- lum serine racemases are actually inhibited by ATP, although the former is activated slightly in the pres- ence of AMP [9,11]. The question hence remains regarding the exact role of the nucleotide in brain mammalian serine racemase catalysis because a PLP-dependent racemization does not require ATP-driven energy. An allosteric role may provide an explanation. In fact, homology modeling indicates that the nucleotide is positioned in the mono- mer ⁄ monomer interface [12 and Fig. 2]. In contrast to the case of E. coli Thr dehydratase mentioned above, we were unable to observe changes in the oligomeriza- tion state of recombinant mouse serine racemase in the presence and absence of added ATP [12]. In fact, all the recombinant versions of serine racemase mentioned above are either homodimers [7–9] or homotrimers [11] in the absence of the nucleotide. Consequently, it is unlikely that ATP might be regulating the quaternary structure of serine racemase. In this regard, as noted by Wolosker and coworkers [4] ATP is not hydrolyzed during catalysis, because both ADP and a nonhydro- lyzable analog of ATP are able to activate the enzyme to a similar extent. Furthermore, in the cytosol the ATP concentrations are in the 3–6 mm range, an obser- vation suggesting that serine racemase is always satu- rated with enough nucleotide to exert its racemase activity because 100 lm ATP is more than enough to result in full activity [4,6]. Perhaps it is even more important to know if serine racemase is activated by Ca 2+ or Mg 2+ in vivo.In principle, if the Mg 2+ concentration in the cellular cytosol is indeed 600 lm [4,16], the brain serine race- mase would always be ‘on’. However, when type II astrocytes were loaded with radioactive d-serine, its release would be induced by l-glutamate and kainate, agents known to increase intracellular calcium concen- trations [17]. Subsequently, we observed the increased release of d -serine by primary astrocytes when gluta- mate, kainate or the calcium ionophore A23187 was added to the cellular medium [5]. Likewise, C6 glioma cells increased their secretion of d-serine when incubated with a-amino-3-hydroxy-5-methylisoxazole- 4-propionic acid (AMPA) [18]. A more direct demon- stration has been recently performed by Mothet and coworkers when they showed that d-serine release is directly related to the increase of cytosolic calcium concentration ([Ca 2+ ] cyt ) [19]. These authors showed that the removal of extracellular calcium, or the deple- tion of thapsigargin-sensitive intracellular calcium stores, abrogated the release of d-serine [19]. It is conceivable that perhaps the increase of [Ca 2+ ] cyt is only involved in the secretion of d-serine previously accumulated in secretion granules [19] although the storage of d-serine in granules in glia has recently been ruled out [18]. It is very likely that serine racemase at various intracellular localizations might be challenged with different calcium concentrations, hence regulating its enzymatic activity. For instance, direct coupling of serine racemase to the AMPA receptor (AMPAR) via glutamate receptor interacting protein (GRIP) binding might be one way to regulate its d-serine synthesizing activity (see below). Nitrosylation of serineine racemase Only scant data are available on possible post-transla- tional modifications of serine racemase in vivo. The observation that both oxidized glutathione [1,5] and cys- tamine [5] could inhibit serine racemase provided some evidence that reactive cysteine residues should be pres- ent that are essential for serine racemase function. When we tested if the nitric oxide ( • NO) donor DETA NONOate [(z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium,2-diolate] could alter serine race- mase activity, we obtained a negative result [5]. Quite recently, reports of • NO as an inhibitor of serine race- mase in a glioblastoma cell line added a new aspect to d-serine-dependent modulation of the glutamatergic synapse. The authors propose that NMDAR-mediated calcium entry into postsynaptic neurons entails cal- cium ⁄ calmodulin-dependent activation of neuronal nitric oxide synthase and the consequent liberation of • NO. Serine racemase is subsequently nitrosylated and inhibited, whereas d-amino oxidase, which is thought to ACP PLP ACP PLP Ca 2+ Ca 2+ Fig. 2. Molecular model of human serine racemase, as described by Baumgart et al. [12]. The calcium ions are depicted as yellow spheres, the PLP moiety is shown in blue and the nucleotide analo- gue phosphomethylphosphonic acid adenylate ester (AMP-PCP) is shown in magenta. The molecular modelling was performed using the crystal structures of the S. pombe serine racemase and the E. coli Thr deaminase. F. Baumgart and I. Rodrı ´ guez-Crespo Biochemistry of brain serine racemase FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS 3541 counteract serine racemase activity in vivo by degrada- tion of d-serine, is upregulated by • NO [20,21]. Subse- quent biochemical proof for this model was provided [22], pinning down the residue that becomes modified and proposing a structural model for the action of • NO. Apparently, cysteine 113 (out of seven cysteine residues in the mouse and human serine racemase sequence) can become nitrosylated, both in the recombinant enzyme and in transfected cells, using the • NO donor, S-nitroso- glutathione (GSNO). A molecular model of mouse ser- ine racemase, based on a yeast homolog, reveals that residue 113 lies in proximity to the putative ATP-bind- ing region of the enzyme. Nitrosylation would therefore lead to impaired nucleotide binding and inactivation of the enzyme. It is noteworthy that GSNO is known to modify cysteines not only with • NO but also with gluta- thione, leading to protein glutathionylation, another post-translational modification occurring under condi- tions of oxidative ⁄ nitrosative stress. In fact, GSNO is very frequently used in glutathionylating studies [23]. It is thus conceivable that purified serine racemase becomes modified by glutathione together with • NO. Experiments with milder nitrosylating reagents that lack a glutathione moiety would unambiguously demon- strate if serine racemase is, in fact, modified by • NO. Serine racemase-interacting proteins: GRIP, PICK1 and Golga3 The carboxy-terminal end of both mouse and human serine racemase display a -Val-serine-Val-COOH sequence, a motif reminiscent of the type II consensus sequence for binding to PSD95 ⁄ disc large ⁄ ZO-1 (PDZ) domains [24]. PDZ domains are among the most ubiquitous protein–protein interaction motifs in metazoan genomes and are especially important in the nervous system for the assembly of synaptic complexes and scaffolding [25,26]. After performing a yeast two- hybrid screen of serine racemase against a rat hippo- campus and cortex cDNA library, the hepta-PDZ protein GRIP was identified as a binding partner of serine racemase [18]. Out of the seven consecutive PDZ modules found in GRIP, serine racemase was found to bind specifically to the PDZ6 domain by means of its C-terminal PDZ-binding motif. Previ- ously, GRIP had been described to interact with gluta- mate receptors of the AMPA ⁄ kainate type [27], where it is responsible for proper trafficking and assembly of the receptor and accessory proteins. GRIP can bind to the glutamate receptor subunit 2 (GluR2) subunit of AMPA receptors via PDZ4 ⁄ PDZ5, both PDZ domains working in concert to establish binding [27,28]. The finding of serine racemase interacting with the PDZ6 domain of GRIP and being activated was the first report on cellular interaction partners of serine race- mase and it raised several intriguing questions. It was not clear whether GRIP directly activated serine race- mase or if binding led to a translocation to the prox- imity of AMPARs in vivo (Fig. 3). Furthermore, the influence of the other PDZ domains of GRIP was not investigated. Therefore, other proteins that become associated with GRIP, using some of the other six PDZ domains, might modulate the activity of serine racemase. Conversely, d-serine might also change the activity of some GRIP-associated proteins. When ser- ine racemase ⁄ GRIP interactions were first studied, it was proposed that GRIP was released from AMPARs when they became stimulated and phosphorylated [18], which would lead to GRIP interacting with serine racemase in the cytosol where it would bind to and activate serine racemase. With our own results we were able to confirm the interaction of GRIP with serine racemase via PDZ6 [12]. However, we observed that binding to PDZ6 alone was not sufficient for activa- tion. Rather, the presence of the rest of the C-terminal region of GRIP, that is the PDZ7 module and a link- ing segment between PDZ6 and PDZ7, was required for full activation of serine racemase, both in vitro and in vivo. Although these results do not necessarily pre- clude a translocation process to AMPARs mediated by GRIP, they do show the direct activation of serine racemase by GRIP as a result of the concerted inter- action of several PDZ modules, independent of the subcellular localization. This PDZ crosstalk, where an isolated PDZ domain is insufficient to carry out a specific function, has also been observed in other examples, for instance in the requirement of both PDZ4 and PDZ5 for GRIP binding to GluR2 [27,28]. Interestingly, the activating effect of GRIP on serine racemase results mainly in a change in V max . More- over, the response curve to calcium remains unchanged upon binding to GRIP under the experimental condi- tions applied, which indicates that GRIP binding and regulation by calcium must be regarded as independent regulation pathways. It has been proposed that serine racemase activation by GRIP takes place in the cytosol after AMPAR phosphorylation and concomitant dis- sociation of GRIP [18]. However, because nothing is known about the kinetics of this process, the forma- tion of a ternary complex among the GluR2 subunit of the AMPA receptor, GRIP and serine racemase cannot be discounted. It would be plausible that GRIP brings serine racemase in close proximity to the gluta- mate-activated channel, where serine racemase might be close to other calcium channels. Although the AMPAR is not a calcium channel, it is conceivable Biochemistry of brain serine racemase F. Baumgart and I. Rodrı ´ guez-Crespo 3542 FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS that in certain calcium microdomains serine racemase could become exposed to temporarily high calcium concentrations. To shut the system off, GluR2 could become phosphorylated, in order to release GRIP and serine racemase, abolishing the transient activation of serine racemase by calcium (Fig. 3). Because GRIP can bind to serine racemase, both in the presence and absence of calcium, it is possible that some other GRIP-interacting protein that also binds to PDZ6 might disrupt the serine racemase–GRIP interaction, hence diminishing the activity of the former. In a similar yeast two-hybrid screen using a human hippocampal cDNA library, a different PDZ domain- containing protein was found to interact with serine racemase, also requiring the C-terminal binding motif [30]. Protein interacting with C kinase 1 (PICK1) con- tains one PDZ domain that is required for interacting with protein kinase C (PKC) [30,31] or serine racemase. It also contains a Bin ⁄ amphiphysin ⁄ Rys domain, important for the interaction with lipids, and a coiled- coil domain. Furthermore, it has been shown recently that the PDZ domain of PICK1 is also capable of inter- acting with lipid membranes, a property crucial for the clustering of AMPAR and synaptic plasticity [32]. There are no data available regarding the effect of the binding of PICK1 on serine racemase activity. There- fore, biochemical characterization of the role of the interaction of serine racemase and PICK1 is needed to judge the importance of these observations. Surely the interaction of PICK1 with PKC leads to the temptation to speculate on a possible phosphorylation of serine racemase by PKC [33]. As yet, however, there are no data available, either on the details of the interaction of PICK1 with serine racemase, or on the phosphorylation of serine racemase. Considering that the phosphorylation of Ser880 of the GluR2 subunit of the AMPA receptor, positioned at the carboxy-terminal end of the polypeptide chain, disrupts its interaction with PDZ4 ⁄ PDZ5 of GRIP, it is tempting to speculate that phosphorylation of Ser336 of human serine racemase or of Thr336 of mouse serine racemase might also break their inter- action with PDZ6 of GRIP. This putative phosphory- latable residue is located at position -3 of the human (Ser–Val–Ser–Val-COOH) and mouse (Thr–Val–Ser– Val-COOH) sequences, respectively, and both are inserted within amino acid sequences of type II con- sensus PDZ domain-interacting partners [24,26]. It has been proposed that PKCa phosphorylates serine race- mase, probably brought into its proximity by PICK1 binding [33]. This hypothesis would rationalize a novel mode of regulation of d-serine synthesis through the activation of nonphosphorylated serine racemase by the multi-PDZ domain GRIP. We have been unable to identify PKCa as a kinase that modifies purified recombinant serine racemase (unpublished data) although perhaps this might be the case in vivo.In addition, both the rat and cow serine racemases are GluR2 AMPAR GluR2 AMPAR GluR2 AMPAR GluR2 AMPAR L-Ser D-Ser C C C C C L-Ser D-Ser C C P C PP P AB C D L-Ser D-Ser L-Ser D-Ser Fig. 3. Proposed modes of interaction among serine racemase, GRIP and the AMPAR. (A) A trimeric complex is assumed. (B) Phosphorylation of the GluR2 subunit of the AMPAR at Ser880 dissociates GRIP binding, which remains bound to serine racemase. (C) Serine racemase is active in the cytoplasm and does not interact with GRIP, whereas the latter associates with the GluR2 subunit. (D) The simultaneous phosphorylation of the GluR2 subunit of the AMPAR together with the phosphorylation of serine racemase releases GRIP to the cytoplasm in the absence of any dual inter- action. F. Baumgart and I. Rodrı ´ guez-Crespo Biochemistry of brain serine racemase FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS 3543 truncated in the carboxy-terminal end, hence lacking GRIP-interacting sequences. Further experiments will demonstrate if the activation of serine racemase by GRIP and its binding to PICK1 is exclusively present in certain mammals or if there is sequence splicing at this region and both rat and cow do have longer (as-yet unidentified) versions of serine racemase. Consequently, at least four different modes of inter- action can be envisaged among AMPAR, serine race- mase and GRIP (Fig. 3). Although phosphorylation of the GluR2 subunit of the AMPAR and the disruption of its association with GRIP have been unambiguously demonstrated, the putative carboxy-terminal phosphor- ylation of serine racemase remains to be established. If GRIP brings serine racemase towards the proximity of the calcium channel, a theoretical modulation of the synthesis of d-serine by calcium concentration can be postulated, in accordance with recent data [18]. Using a mouse brain lysate we observed a trimeric GluR2– GRIP–serine racemase (data not shown) although we do not know which mechanisms lead to the dissocia- tion of serine racemase from GRIP. Nevertheless, in the absence of the association with the AMPAR, recombinant purified GRIP alone is able to increase the activity of recombinant purified serine racemase [12]. We have shown that certain GRIP amino acids, present further down in the sequence than PDZ6, are responsible for the majority of the observed activation of serine racemase by GRIP [12]. In another study to identify binding partners of ser- ine racemase using the yeast two-hybrid technology, the Golgi-localized protein, Golgin subfamily A mem- ber 3 (Golga3), was found to interact with serine race- mase [34]. In this case, however, no PDZ interactions with the C-terminal amino acid triplet of serine were crucial for binding, but instead, the interaction was established with its N-terminal 66 residues. Binding of Golga3 increases d-serine synthesis. Intriguingly, this is achieved through a decrease in ubiquitin ⁄ proteasomal degradation of serine racemase, rather than by modula- tion of the catalytic rate. Serine racemase was shown to have an average half-life of about 4.5 h. When Golga3 and serine racemase were cotransfected, both serine racemase stability and d-serine synthesis increased con- siderably. Thus, it is important to note that in addition to the modulators mentioned beforehand which directly influence the catalysis of serine racemase, indirect effects such as protein stability or subcellular localiza- tion should be taken into account when investigating the precise regulation of serine racemase-dependent d-serine levels at glutamatergic synapses. 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Neuron Glia Biol 1, 275–281. 34 Dumin E, Bendikov I, Foltyn VN, Misumi Y, Ikehara Y, Kartvelishvily E & Wolosker H (2006) Modulation of d-serine levels via ubiquitin-dependent proteasomal degradation of serine racemase. J Biol Chem 281, 20291–20302. F. Baumgart and I. Rodrı ´ guez-Crespo Biochemistry of brain serine racemase FEBS Journal 275 (2008) 3538–3545 ª 2008 The Authors Journal compilation ª 2008 FEBS 3545 . whereas those involved in protein–protein interaction are shown in green. The first four amino acids of the barley serine racemase and the final 88 amino acids of the Pyrobaculum serine racemase are. [32]. There are no data available regarding the effect of the binding of PICK1 on serine racemase activity. There- fore, biochemical characterization of the role of the interaction of serine racemase. domain- containing protein was found to interact with serine racemase, also requiring the C-terminal binding motif [30]. Protein interacting with C kinase 1 (PICK1) con- tains one PDZ domain that