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At least 19 recordsLinked to original sources

PubMed2004

Serine racemase and D-serine transport in human placenta and evidence for a transplacental gradient for D-serine in humans.

OBJECTIVE: To investigate the possible role of human placenta in providing D-serine to the developing fetus. METHODS: Expression of serine racemase in placenta was determined by reverse transcriptase polymerase chain reaction and northern analysis and confirmed by subsequent cloning. The transport of D-serine by human ATB(0) was characterized by expressing the cloned cDNA transiently in mammalian cells using the vaccinia virus expression system. D-serine levels in maternal and fetal blood were measured by fluorescence high-performance liquid chromatography (HPLC) after derivatization of the amino acids with o-phthaldialdehyde and N-tertiary-butyloxycarbonyl-L-cysteine. RESULTS: mRNA for serine racemase was detected in placenta. ATB(0) was capable of d-serine transport, and the transport process is obligatorily dependent on sodium (Na+) with a Na(+):substrate stoichiometry of 1:1 and saturable with a Michaelis-Menten constant of 310 +/- 30 microM. Furthermore, studies have shown that ATB(0) is not expressed in the maternal-facing brush border membrane of human placental syncytiotrophoblast. The circulating concentration of D-serine in maternal serum is 5.8 +/- 0.5 microM, and the corresponding value in the fetal serum is 14.6 +/- 1.2 microM, indicating a two- to three-fold higher concentration of D-serine in the fetus than in the mother. CONCLUSION: We speculate that D-serine is synthesized in human placenta by the racemization of L-serine and that ATB(0), expressed on the basal membrane of the syncytiotrophoblast, mediates the efflux of D-serine into the fetal circulation in exchange for other amino acids in fetal blood.

Biological Transport↗

PubMed2004

Reactions of serine palmitoyltransferase with serine and molecular mechanisms of the actions of serine derivatives as inhibitors.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395(T) in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5'-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to L-serine and product, and showed the following new aspects: First, we analyzed the binding of L-serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various serine analogues were also examined; the data indicated that the alpha-carboxyl group of L-serine was quite important for substrate recognition by SPT. Third, we focused on a series of SPT inhibitors, which have been used as convenient tools to study the cell responses caused by sphingolipid depletion. The interaction of SPT with myriocin suggested that such product-related compounds would strongly and competitively inhibit enzyme activity by forming an external aldimine in the active site of the enzyme. Beta-chloro-L-alanine and L-cycloserine were found to generate characteristic PLP-adducts that produced inactivation of SPT in an irreversible manner. The detailed mechanisms for the SPT inactivation were discussed. This is the first analysis of the inhibition mechanisms of SPT by these compounds, which will provide an enzymological basis for the interpretation of the results from cell biological experiments.

Acyltransferases↗

PubMed2000

Dexamethasone increases the incorporation of [3H]serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: a possible relation between serine base exchange enzyme and apoptosis.

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [3H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.

Aluminum Compounds↗

PubMed2000

Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

Amino Acid Sequence↗

PubMed1998

Evidence of a functional interaction between serine 3 and serine 25 Mos phosphorylation sites. A dominant inhibitory role of serine 25 phosphorylation on Mos protein kinase.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.

3T3 Cells↗

PubMed2002

Effect of the intracerebroventricular and systemic administration of L-serine on the concentrations of D- and L-serine in several brain areas and periphery of rat.

To gain further insight into the metabolic mechanism of endogenous D-serine, the effect of the intracerebroventricular and intraperitoneal administration of L-serine on the concentrations of D- and L-serine in several brain areas and periphery was investigated. The intracerebroventricular injection of L-serine caused a rapid and marked increase in the L-serine levels in almost all brain regions of adult rats. This administration also produced a gradual increase in the D-serine levels in the forebrain, whereas a slight but significant elevation of D-serine was found in the cerebellum and pons-medulla. The intraperitoneal administration of L-serine caused a marked increase in the L-serine levels in all brain regions of both infant and adult rats. The treatment induced a significant augmentation of the D-serine levels in all brain regions of infant rats with higher concentrations in the cerebellum and cortex, whereas no significant change was observed in the cerebellum and pons-medulla of adult rats. These in vivo observation, together with the fact that immunohistochemical studies have indicated that both D-serine and serine racemase are highly concentrated in Bergmann glia of developing cerebellum, suggest that D-serine can be synthesized not only in the forebrain but also in the hindbrain by serine racemase. Furthermore, because the drastic decline in the cerebellar D-serine level coincides well with a dramatic increase in the cerebellar D-amino acid oxidase during early postnatal development, synthesized D-serine may be metabolized by D-amino acid oxidase in the hindbrain of adult rats.

Aging↗

PubMed1997

Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.

Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation; this is a major phenomenon since together these metabolites represent 60% of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 microM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 microM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 microM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not into Etn. Increasing exogenous serine to 100 microM decreased the incorporation of radioactive Etn into PtdEtn by only 30%, and the PtdCho level was not affected. 2-Hydroxyethylhydrazine significantly decreased serine incorporation into P-Etn and PtdEtn, whereas Etn was accumulated. No concomitant inhibition of PtdSer or PtdCho labelling from serine occurred, even when PtdEtn formation was decreased by 95%. This indicates that the PtdEtn pool derived from direct serine decarboxylation differed from that derived from PtdSer decarboxylation, and the latter appeared to be preferentially used for PtdCho biosynthesis. Hydroxylamine also inhibited phosphorylation of serine-derived Etn but not that of exogenous Etn. The rate of PtdSer synthesis from 10 microM L-serine was 3.1+/-0.5 and 2.95+/-1.3 nmol/5 h per 10(10) infected cells, whereas L-serine decarboxylation accounted for 7.1+/-1.5 and 9.9+/-3 nmol/5 h per 10(10) infected cells for P. falciparum and P. knowlesi respectively (means+/-S.E.M.). The serine decarboxylating reaction was not detected in other higher eukaryotic cells such as mouse fibroblasts and human lymphocytes. Finally, these results also indicate compartmentalization of phospholipid metabolism in Plasmodium-infected erythrocytes.

Animals↗

PubMed1995

Studies into the identity of the sites of insulin-stimulated insulin receptor serine phosphorylation. Characterization of synthetic peptide substrates for the insulin-stimulated insulin receptor serine kinase.

The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin receptor copurified with insulin-stimulated insulin receptor serine kinase activity. Of the 14 peptides studied, only 4 (1071--1080, 1290--1298, 1253--1271, and 1313--1329) were phosphorylated on serine, with the serine phosphorylation stimulated 2--4-fold by insulin. Peptides 1071--1080 and 1290--1298 were 3--7-fold better substrates for the serine phosphorylation than the other serine-phosphorylated peptides. Peptides 1071--1080 and 1313--1329 also exhibited insulin-stimulated phosphorylation on tyrosine. Two-dimensional thin-layer tryptic mapping of the phosphorylated insulin receptor/insulin-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptides, called S1 and S2, that ran as a pair of closely migrating spots, and other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosposerine/phosphotyrosine ratios of approximately 6 and 0.96-1.50 for the in vivo and in vitro labeled receptor, respectively. S1 and S2 were not cleaved by V8. Of the serine-phosphorylated peptides, only 1290--1298 and 1071--1080 should be V8 resistant; 1290--1298 contains serine sites 1293/4 and migrated distinctly from S1 and S2 in tryptic maps. Peptide 1071--1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphopeptides, each containing phosphoserine and phosphotyrosine, which comigrated exactly with S1 and S2. Comigration was confirmed at a different pH and by mixing experiments. Radiosequenation showed that serine 1078 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it was no more than a minor site in vivo. It is concluded that serine 1078 of the insulin receptor is a major site of insulin-stimulated phosphorylation in vivo and in vitro. The peptide sequences provide a range of substrates to facilitate the study, purification, and characterization of the insulin-stimulated insulin receptor serine kinase or kinases, and the identification of a major site of insulin-stimulated serine phosphorylation will help elucidate the function of the insulin receptor serine phosphorylation.

Amino Acid Sequence↗

PubMed2001

A new strategy to decrease N-methyl-D-aspartate (NMDA) receptor coactivation: inhibition of D-serine synthesis by converting serine racemase into an eliminase.

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Ammonia↗

PubMed2003

Spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase during mouse postnatal development.

AIM: To elucidate the spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase (EC 1.4.3.3; DAO) in mouse cortex, striatum, cerebellum, heart, lung, liver, spleen, kidney, and skeletal muscle during mouse postnatal development. METHODS: The transcription levels of serine racemase and DAO were assayed by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of serine racemase were examined by Western blot. DAO activities were assayed by colorimetric method. D-serine was measured by HPLC. RESULTS: In cortex, striatum, and cerebellum, free D-serine increased drastically after birth and coincided well with the increase of serine racemase expression. However, among the 9 tissues examined, DAO activities were detected only in cerebellum and kidney. During the 3rd week, DAO activity in cerebellum and kidney increased dramatically, which concurred with the drastic decline of D-serine content in these tissues. On the other hand, while D-serine and serine racemase fall to trace level in cerebellum and kidney at the 3rd weekend, DAO activities in these tissues increased continuously. CONCLUSION: The free D-serine is mainly synthesized by serine racemase. However, novel mechanisms might be involved in D-serine deposition in mouse tissues with high level of D-serine and no detectable DAO activity such as cortex and striatum. DAO in cerebellum and kidney might have other physiological functions in addition to degrading D-amino acid.

Animals↗

PubMed1989

In vitro conversion of formate to serine: effect of tetrahydropteroylpolyglutamates and serine hydroxymethyltransferase on the rate of 10-formyltetrahydrofolate synthetase.

Serine hydroxymethyltransferase and C1-tetrahydrofolate synthase catalyze four reactions which convert formate and glycine to serine. The one-carbon carrier in these reactions if tetrahydropteroylglutamate which is regenerated in the coupled reaction and thus can be used in catalytic concentrations with respect to serine synthesis. The rate of serine synthesis is followed by the oxidation of NADPH during reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. Km values for the substrates of cytosolic serine hydroxymethyltransferase and the 10-formyltetrahydrofolate synthetase activity of the trifunctional enzyme C1-tetrahydrofolate synthase were determined. This included the values for the polyglutamate forms of tetrahydropteroylglutamate containing from one to six glutamate residues. The results suggest that the synthetase active site binds the polyglutamate forms of the coenzyme synergistically with respect to formate and ATP. Using saturating levels of all substrates, the kcat values for the serine hydroxymethyltransferase and 10-formyltetrahydrofolate synthetase activities were also determined. The synthetase reaction is the rate-determining step in the conversion of formate to serine. The effect of glutamate chain length and the concentration of serine hydroxymethyltransferase were studied with respect to the rate of serine formation. Tetrahydropteroylmonoglutamate gave slower than expected rates which is attributed to its inhibition of the reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. This inhibition was not a factor with the di- through hexaglutamate forms of the coenzyme. The addition of an excess of serine hydroxymethyltransferase was predicted to lower the rate of the formation of serine by lowering the concentration of free coenzyme in the assay. However, activation of the rate was observed which was at least 2-fold greater than the predicted rate. This increase in predicted rate appears to result from an interaction between C1-tetrahydrofolate synthase and serine hydroxymethyltransferase. The in vivo concentrations of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in rabbit liver were determined.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1985

Kidney independent serine synthesis in the rat. Effect of dietary serine and glycine restriction.

The potential role of the kidney in renal serine synthesis was elucidated in rats following dietary serine as well as serine and glycine depletion for 6 weeks. Serine deficient diet caused a significant rise in blood serine and glycine values. In contrast, blood glycine concentration decreased significantly following serine and glycine depletion, whereas blood serine levels remained unchanged. Bilateral nephrectomy caused a significant decrease of blood serine values of control and serine deficient rats. Serine concentration, however, was not affected by bilateral nephrectomy following serine and glycine deficiency indicating kidney-independent serine synthesis under these conditions. There was a significant increase of blood lactate and pyruvate levels under serine and serine/glycine deficient diet compared with control animals, whereas blood ketone bodies fell. In contrast, 24 h after bilateral nephrectomy blood ketone bodies rose and blood lactate and pyruvate concentrations decreased significantly. 24 h after bilateral nephrectomy there was a marked rise in blood citrate values of female rats compared with male animals indicating sex dependency of kidney citrate metabolism in the rat.

3-Hydroxybutyric Acid↗

PubMed1999

Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.

Adenosine Triphosphate↗

PubMed2004

L-serine catabolism via an oxygen-labile L-serine dehydratase is essential for colonization of the avian gut by Campylobacter jejuni.

Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of L-serine from a defined medium containing a mixture of amino acids led to a dramatic decrease in cell yields. As C. jejuni does not have a biosynthetic serine requirement, this supports earlier suggestions that L-serine is a preferentially catabolized amino acid. Serine transport was found to be mediated by at least two systems in strain 11168; a high-capacity, low-affinity L-serine-specific system encoded by Cj1625c (sdaC) and a higher-affinity L-serine/L-threonine system responsible for residual L-serine transport in an sdaC mutant. Catabolism of L-serine to pyruvate and ammonia is carried out by SdaA (encoded by Cj1624c), which was overexpressed, purified, and shown to be an oxygen-labile iron-sulfur enzyme. L-Serine dehydratase activity in an sdaA mutant was reduced 10-fold compared to that in the wild type, but the residual activity (due to the anabolic L-threonine dehydratase) could not support either growth on or utilization of L-serine in defined media. However, although sdaA mutants showed no obvious growth defect in complex media, they completely failed to colonize 3-week-old chickens as assayed both by cloacal swabs taken over a 6-week period and by cecal colony counts postmortem. In contrast, the isogenic parent strain colonized chickens to high levels within 1 week of inoculation. The results show that an active SdaA is essential for colonization of the avian gut by C. jejuni and imply that catabolism of L-serine is crucially important for the growth of this bacterium in vivo.

Animals↗

PubMed1994

The primary structure of sheep liver cytosolic serine hydroxymethyltransferase and an analysis of the evolutionary relationships among serine hydroxymethyltransferases.

The complete amino-acid sequence of sheep liver cytosolic serine hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver serine hydroxymethyltransferase consisted of 483 amino-acid residues. A comparison of this sequence with 8 other serine hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the serine hydroxymethyltransferases belong to the alpha/beta category of proteins. The predicted secondary structure of sheep liver serine hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5'-phosphate containing enzyme, suggesting that sheep liver serine hydroxymethyltransferase might have a similar pyridoxal 5'-phosphate binding domain. In addition, a conserved glycine rich region, G L Q G G P, was identified in all the serine hydroxymethyltransferases and could be important in pyridoxal 5'-phosphate binding. A comparison of the cytosolic serine hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that serine hydroxymethyltransferase was a highly conserved protein. It was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5'-phosphate against modification with [14C]iodoacetic acid, while C247 and C261 were buried in the native serine hydroxymethyltransferase. However, the cysteines are not conserved among the various serine hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by serine hydroxymethyltransferase remains to be elucidated.

Amino Acid Sequence↗

PubMed2004

Induction of serine racemase expression and D-serine release from microglia by amyloid beta-peptide.

BACKGROUND: Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid beta-peptide (Abeta), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly. METHODS: Cultured microglial cells were exposed to Abeta. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase. RESULTS: Conditioned medium from Abeta-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Abeta elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx). In the microglia, Abeta elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Abeta. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls. CONCLUSIONS: These data suggest that Abeta could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine.

Journal Article↗

PubMed1974

Regulation of hepatic L-serine dehydratase and L-serine-pyruvate aminotransferase in the developing neonatal rat.

1. The activities of l-serine dehydratase and l-serine-pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine-pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine-pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action.

Animals↗

PubMed1994

Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability.

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an alteration in the apparent Tm of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 +/- 5) x 10(-3) s-1 and (69 +/- 7) x 10(-3) s-1 for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme.

Animals↗