The 1986 Borden award lecture. The role of the kidney in amino acid metabolism and nutrition

Measurement of the arteriovenous differences for free amino acids across rat kidney reveals that glycine and citrulline are removed and serine and arginine are added to the circulation. In addition, glutamine is taken up in large quantities by kidneys of animals that need to excrete large quantities of acid (e.g., diabetic animals, NH4Cl-fed animals, and animals fed a high protein diet). Glutamine is the major precursor of urinary ammonia and thus renal glutamine metabolism plays a key role in acid-base homeostasis. This process occurs primarily in the cells of the convoluted proximal tubule. Glutamine carbon is converted to glucose in acidotic rats and is totally oxidized in dogs. Regulation of glutamine metabolism occurs at two levels: acute regulation and chronic regulation. Acute regulation is, in part, mediated through a fall in intracellular [H+]. This activates alpha-ketoglutarate dehydrogenase and, ultimately, glutaminase. Chronic regulation involves induction of key enzymes, including, in the rat, glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase. During the acidosis of prolonged starvation, the kidneys' requirement for glutamine must be met from muscle proteolysis and thus becomes a drain on lean body mass. Serine synthesis occurs by two separate pathways: from glycine by the combined actions of the glycine cleavage enzyme and serine hydroxymethyltransferase and from gluconeogenic precursors using the phosphorylated-intermediate pathway. Both pathways are located in the cells of the proximal tubule. Conversion of glycine to serine is ammoniagenic and the activity of the glycine cleavage enzyme is increased in acidosis. The function of serine synthesis by the phosphorylated-intermediate pathway is not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.     

Reprogramming of serine,glycine and one-carbon metabolism in cancer

Metabolic pathways leading to the synthesis, uptake, and usage of the nonessential amino acid serine are frequently amplified in cancer. Serine encounters diverse fates in cancer cells, including being charged onto tRNAs for protein synthesis, providing head groups for sphingolipid and phospholipid synthesis, and serving as a precursor for cellular glycine and one-carbon units, which are necessary for nucleotide synthesis and methionine cycle reloading. This review will focus on the participation of serine and glycine in the mitochondrial one-carbon (SGOC) pathway during cancer progression, with an emphasis on the genetic and epigenetic determinants that drive SGOC gene expression. We will discuss recently elucidated roles for SGOC metabolism in nucleotide synthesis, redox balance, mitochondrial function, and epigenetic modifications. Finally, therapeutic considerations for targeting SGOC metabolism in the clinic will be discussed.     

Mitochondrial cysteine synthase complex regulates o-acetylserine biosynthesis in plants

Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [(3)H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand.     

Serine utilization in mouse liver: influence of caloric restriction and aging

The influence of caloric restriction (CR) on the activities of hepatic serine metabolizing enzymes in young (3 months) and old (30 months) mice was studied. Serine dehydratase (SDH) activity increased markedly with age in both diet groups and in old mice was higher in the CR group. No effects of CR were observed in the young. Serine:pyruvate transaminase (SPT) and glycerate kinase activities were unaffected by age and diet. However, glycerate dehydrogenase activity was decreased in old CR mice but not in young CR. The results of this study show that long-term CR influenced serine utilization only in the pathway catalyzed by SDH. This suggests that in mouse liver this pathway is critical for serine utilization in gluconeogenesis, while the SPT pathway plays a minor role. The increase in SDH activity with long-term CR is consistent with sustained increase in gluconeogenesis.     

l-Serine transport in growing and maturing mouse oocytes

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis–Menten constant (K_m) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus–oocyte complexes.     

Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.     

Nuclear Compartmentalization of Serine Racemase Regulates d-Serine Production: IMPLICATIONS FOR N-METHYL-d-ASPARTATE (NMDA) RECEPTOR ACTIVATION*

d-Serine is a physiological co-agonist that activates N-methyl d-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. d-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. d-Serine is synthesized by the enzyme serine racemase (SR), which directly converts l-serine to d-serine. However, many aspects concerning the regulation of d-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of d-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular d-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.     

One-carbon metabolism in lectin-activated human lymphocytes

Serine is an essential amino acid for the lectin-mediated transformation of human peripheral blood lymphocytes due to the inability of this cell to synthesize sufficient quantities via either the phosphorylated pathway or by reversal of the serine hydroxymethyltransferase reaction to meet the metabolic demands. The level of intracellular serine is tightly regulated, and the culture medium concentration for optimum cellular transformation falls within a relatively narrow range. The three-carbon atom of serine is the major source of one-carbon units required for purine and pyrimidine nucleotide biosynthesis, but the key effect of both serine deprivation and of high medium serine levels would appear to be on protein synthesis. Although an alternative source of one-carbon units, as provided by high levels of formate in the culture medium, can partially reverse the effects of serine deprivation, the only other demonstrable source of one-carbon units, tryptophan, requires serine for its incorporation and subsequent metabolism. Methionine is also essential for lymphocyte transformation and is involved in the synthesis of a small amount of phosphatidylcholine, although most of this phospholipid is provided by choline and lysophosphatidylcholine from the serum-supplemented culture medium.     

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1^C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1^C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.     

Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro

We have investigated the functional significance of phosphoserine residues that lie in the L protein-binding domain between amino acids 213 and 247 of the phosphoprotein (NS) of vesicular stomatitis virus. A series of mutant NS proteins were made by cell-free translation of mRNAs transcribed from the cloned gene. Site-directed substitution of alanine for both serine 236 and serine 242 essentially abolished RNA synthesis catalyzed by the NS-L complex. Substitution of either of these serines reduced RNA synthesis by 75%. Serine 218 played no major role in RNA synthesis. Phosphorylation of NS by the L protein was abrogated by substitution of either serine 236 or serine 242. These results indicate that phosphorylation of serines 236 and 242 in the NS protein regulates its binding with the L protein and the N-RNA template and is essential for activation of viral RNA synthesis.     

Serine metabolism in human pregnancy

Serine plays an important role in intermediary metabolism as a source of one carbon pool for nucleotide biosynthesis, as a precursor for glycine and glucose, and as a contributor to cysteine biosynthesis. A unique serine-glycine cycling between the liver and the placenta has been demonstrated in the sheep fetus. We hypothesized that, because of serine's role in growth and development, significant changes in serine metabolism will occur in pregnancy with advancing gestation. The rate of appearance (R(a)) of serine and its metabolism were quantified in healthy women longitudinally through pregnancy with a [2-(15)N(13)C]serine tracer. The contribution of serine N to urea and the rate of oxidation of serine were measured using the precursor-product relation. Plasma serine concentrations and serine R(a) were lower in pregnant (P) women, in both early and late gestation, compared with nonpregnant (NP) women [plasma serine: NP, 113 +/- 24.5; P early, 71.9 +/- 6.2; P late, 68.5 +/- 9.6 micromol/l; serine R(a): NP (n = 7), 152.9 +/- 42.8; P early (n = 12), 123.7 +/- 21.5; P late (n = 8), 102.8 +/- 18.2 micromol x kg(-1) x h(-1)]. Serine contributed approximately 6% to urea N and 15-20% to the plasma glycine pool, and oxidation of serine represented approximately 8% of R(a). There was no significant difference between P and NP subjects. Glucose infusion, at 3 mg x kg(-1) x min(-1) in P subjects, resulted in a decrease in serine R(a) and an increase in oxidation. The decrease in serine turnover in pregnancy may represent a decrease in alpha-amino nitrogen turnover related to a decreased rate of branched-chain amino acid transamination and caused by pregnancy-related hormones aimed at nitrogen conservation and accretion.     

Serine proteases of small intestine mucosa — localization,functional properties,and physiological role

In this review we present data about small intestine serine proteases, which are a considerable part of the proteolytic apparatus in this major part of the gastrointestinal tract. Serine proteases of intestinal epitheliocytes, their structural-functional features, cellular localization, physiological substrates, and mechanisms of activity regulation are examined. Information about biochemical and functional properties of serine proteases is presented in a common context with morphological and physiological data, this being the basis for understanding the functional processes taking place in upper part of the intestine. Serine proteases play a key role in the physiology of the small intestine and provide the normal functioning of this organ as part of the digestive system in which hydrolysis and suction of food substances occur. They participate in renewal and remodeling of tissues, retractive activity of smooth musculature, hormonal regulation, and defense mechanisms of the intestine.     

D-amino acids in the brain: the biochemistry of brain serine racemase

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 alpha,beta-elimination 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 interacting 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.     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.     

Effect of Serine Hydroxamate on Phospholipid Synthesis in Escherichia coli

Serine hydroxamate, which inhibits the charging of seryl-transfer ribonucleic acid, reduced the synthesis of phospholipid and nucleic acids in Escherichia coli. This effect was analogous to depriving amino acid auxotrophs of their nutritional requirement and appears to be a manifestation of the stringent response shown by rel(+) strains of E. coli. Amino acid starvation (serine or methionine) alone or serine hydroxamate treatment alone results in 60 to 80% inhibition of lipid accumulation, 90% inhibition of ribonucleic acid accumulation, and an increase in guanosine tetraphosphate (ppGpp). These three effects were reversed by addition of chloramphenicol (CM). A combination of serine starvation and serine hydroxamate treatment resulted in inhibition of lipid and RNA accumulation as well as an increase in ppGpp, but the consequences of the double block were not reversed by CM. We conclude that a strong interrelationship exists among these processes and that CM acts to relax a stringent response by mechanisms other than interference with ppGpp formation. All species of phospholipid were affected by a stringent response evoked by amino acid starvation or addition of serine hydroxamate, but in all cases the synthesis of phosphatidylethanolamine was most severely inhibited. Serine hydroxamate was not incorporated into lipid but specifically caused phosphatidylserine accumulation. Serine starvation produced a dramatic alteration of the distribution of isotope incorporated into phospholipid, which resulted from the stringent response compounded with the limitation of a substrate for phosphatidylserine synthesis.     

Nitrogen-economy and synthesis of serine and glycine in reticulocytes

Amino acids constitute the main substrate of the reticulocyte. The amino acid pool of reticulocytes represents a characteristic selection. The composition of the amino acid pool is dependent on maturation. From the preferential oxidation of short-chain amino acids one would expect a ratio about of 5:1 between the oxygen consumption and ammonia formation. If one corrects for NH3-formation by the deamination of nucleotides the ratio between the oxygen consumption and NH3-formation is about an order of magnitude higher than the theoretical ratio. The small liberation of NH3 in energy production from amino acids results from the re-utilization of their alpha-NH2-group for the synthesis of serine and glycine, while the C-skeleton stems from glucose. Serine is formed via OH-pyruvate and OH-pyruvate. Serine and glycine serve preferentially for synthesis of hemoglobin. In reticulocytes there exists a compartmentation of glycine which accounts for differences between serine and glycine in isotopic experiments. From the time dependent change of the specific activities of pulse-labelled serine and glycine one may calculate that the serine synthesis amounts to 15--30% of the glucose utilization.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Disruption of thymidylate synthesis and glycine-serine interconversion by L-methionine and L-homocystine in Raji cells

Excessive concentrations of L-methionine inhibited the folate-dependent de novo synthesis of thymidylic acid (TMP) in Raji cells, demonstrating the usefulness of this cell line for the study of methionine-folate antagonism. The effect was also produced by L-homocystine but not by other amino acids including D-methionine and L-ethionine, suggesting that this effect is exerted by a common intermediate of methionine and homocystine metabolism. L-Methionine, L-homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) are not inhibitors of thymidylate synthase activity. On the other hand the capacity of the cells to incorporate serine 3-carbon and glycine 2-carbon into DNA is impaired by the presence of L-methionine or L-homocystine. Studies with cell-free extracts demonstrated that the glycine cleavage enzyme is inhibited by 45% by L-methionine, L-homocysteine, SAM or SAH. Serine hydroxymethylase on the other hand was slightly stimulated by these sulfur-containing compounds and this stimulation was shown to occur in the intact cell as well. These findings suggest that when levels of L-methionine metabolites are elevated, there is an increase in the use of glycine to maintain the intracellular concentration of serine, which is required for homocysteine detoxification by conversion to cystathionine. The reduction in TMP synthesis caused by excess L-methionine or L-homocystine may result from increased utilization of one-carbon units for serine synthesis.