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At least 235 records · Page 13Linked to original sources
PubMed1993

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+.

MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1997

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors.

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.

Journal Article↗
PubMed1984

Enzymes of serine metabolism in normal, developing and neoplastic rat tissues.

The cellular pattern of serine metabolism was conceptualized into four main areas of metabolic sequences: the biosynthesis of serine from intermediates of the glycolytic pathway (the so-called "phosphorylated pathway"); and alternative pathways of serine utilization initiated by serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. The known regulatory and adaptive properties of the enzymes involved in these pathways were reviewed in detail and key enzymes associated with each pathway (phosphoserine aminotransferase, serine dehydratase, serine aminotransferase, and serine hydroxymethyltransferase, respectively) were selected for further investigation. Tissue distribution studies in the rat revealed that whereas serine dehydratase and serine aminotransferase activities were largely confined to the liver, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were more broadly distributed. In particular in tissues with a high rate of cell turnover, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were coordinately increased. An increase in serine hydroxymethyltransferase activity coincided temporally with the incorporation of [3-14C]serine and thymidine into DNA in normal human lymphocytes during proliferation after mitogenic stimulation by phytohemagglutinin. The evidence suggested a primarily gluconeogenic role for serine dehydratase and serine aminotransferase. Serine hydroxymethyltransferase has a role in providing glycine and one-carbon folate co-factors as precursors for nucleotide biosynthesis and in some situations serves to metabolically couple the pathway of serine biosynthesis to utilization for de novo purine and pyrimidine synthesis. Multiple enzymic forms were distinguished for serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. For serine dehydratase the two cytosolic multiple forms had no apparent functional significance; the multiple forms were catalytically unmodified by conditions promoting phosphorylation-dephosphorylation in vitro. The mitochondrial form of serine aminotransferase showed adaptive responses in gluconeogenic situations, and the hypothesis was proposed that the mitochondrial isoenzyme of serine hydroxymethyltransferase is associated together with serine aminotransferase in a pathway for gluconeogenesis from protein-derived amino acids such as glycine and hydroxyproline. The adaptive behaviour of the enzymes during the neonatal development of rat liver revealed that serine aminotransferase reached a peak in the mid-suckling period at a time when gluconeogenesis is known to be increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors.

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.

Animals↗
PubMed2002

Glial transport of the neuromodulator D-serine.

D-Serine is an endogenous agonist of NMDA receptors that occurs in astrocytes in gray matter areas of the brain. D-Serine is synthesized from L-serine by the activity of a glial enriched serine racemase, but little is known on the properties of D-serine transport and factors regulating its synaptic concentration. In the present report we characterize the transport of D-serine in astrocytes. In primary astrocyte cultures, D-serine uptake is dependent on sodium ions and exhibits both low affinity and low specificity for D-serine. The kinetics of D-serine transport resembles that of ASCT type transporters as several small neutral amino acids strongly inhibit the uptake of D-serine. D-Serine fluxes are coupled to counter-movement of L-serine and to a less extent to other small neutral amino acids. Thus, addition of D-serine to cell cultures elicits robust efflux of intracellular L-serine. Conversely, physiological concentrations of L-serine induce efflux of preloaded D-serine from astrocytes. L-Serine was more effective than kainate, which have been previously shown to induce D-serine release from astrocytes upon stimulation of non-NMDA type of glutamate receptors. The features of D-serine transport we describe reveal possible new mechanisms controlling the synaptic concentration of D-serine.

Amino Acids, Neutral↗
PubMed2002

Folate cofactors regulate serine metabolism in fetal ovine hepatocytes.

The fetal liver is the primary site of fetal serine production. The regulation of this unique fetal hepatic serine production is unknown. We hypothesized that serine production would be responsive to folate cofactor supply or hormonal regulation. To test this hypothesis, we determined the effect of key folate cofactors and insulin and glucagon on serine and glycine metabolism in primary culture of fetal ovine hepatocytes. Hepatocytes were cultured in serum-free, low-folate media [5 nM 5-methyl-tetrahydrofolate (THF)] with or without 50 nM 5,10-methylene-THF (MTHF) or 5-formyl-THF (FTHF). Serine and glycine production (P) and utilization (U) were determined by stable isotope dilution with [1-(13)C]serine and [1-(13)C]glycine for 24 h. The effect of insulin (1 microM) or glucagon (1 micro M) was determined in a similar manner. Under basal conditions, serine P (43.2 +/- 5.1 micromol/mg DNA per 24 h) is greater than serine U (24.1 +/- 3.1 micromol/mg DNA per 24 h), whereas glycine U (27.3 +/- 3.0 micromol/mg DNA per 24 h) exceeds glycine P (16.7 +/- 1.9 micromol/mg DNA per 24 h). MTHF results in a significant decrease in serine U (16.0 +/- 2.7 micromol/mg DNA per 24 h; p = 0.02 versus low folate), with no change in serine P. FTHF reduces serine P (36.2 +/- 4.9 micromol/mg DNA per 24 h; p = 0.01), but does not alter serine U. There were no effects on glycine metabolism with 50 nM MTHF or FTHF. Serine P and U were inversely correlated whereas glycine P and U were directly correlated with the media concentration of MTHF or FTHF. Glucagon treatment increased serine U by 260 +/- 65% versus low folate (p = 0.0004) but did not change serine P. Insulin treatment led to parallel increases in both serine P and U. Both folate cofactor availability and hormone concentrations regulate serine metabolism in the fetal liver. We speculate that serine metabolism may be a marker of fetal hepatic folate cofactor supply.

Animals↗
PubMed1996

Serine and glycine metabolism in hepatocytes from mid gestation fetal lambs.

Using stable isotopes of serine, glycine, and glutamine, the metabolism of serine and glycine was investigated in primary hepatocytes from six mid-gestation fetal lambs (mean gestational age = 81 +/- 6 d, normal gestation = 145 d). Serine production was 6.84 +/- 1.22 mumol/24 h/mg of DNA and exceeded serine utilization (3.76 +/- 1.44 mumol/24 h/mg of DNA) with a resultant net increase in medium serine of 2.58 +/- 1.70 mumol/24 h/mg of DNA. In contrast, glycine production (6.84 +/- 1.16 mumol/24 h/mg of DNA) was less than glycine utilization (12.10 +/- 1.78 mumol/24 h/mg of DNA) with a net decline in medium glycine of -5.44 +/- 2.03 mumol/24 h/mg of DNA. Of the serine produced, 50.4 +/- 4.3% was derived from glycine via the action of serine hydroxymethyltransferase (SHMT) and the glycine cleavage enzyme complex (GCS). Increasing the medium serine concentration resulted in an increase in serine utilization and sparing of the utilization of other amino acids. Biosynthesis of glycine from serine accounts for only 18.1 +/- 5.6% of glycine production, and this percentage is not affected by changes in medium serine concentration. Using 2.5-[15N2]glutamine as the tracer, an estimated 18 +/- 7% of serine production was derived from transamination reactions. The specific activity of both cytosolic and mitochondrial SHMT was constant for the duration of the cultures. We conclude that, in mid-gestation fetal ovine hepatocytes, there is net production of serine (with glycine as the primary metabolic source of this serine biosynthesis) and net glycine utilization. These data suggest that flux through SHMT and GCS accounts for 50% of serine biosynthesis in mid-gestation fetal ovine hepatocytes. The sparing of the utilization of other amino acids by serine suggests that serine a conditionally essential amino acid for the mid-gestation fetal liver.

Amino Acids↗
PubMed2004

Chiral enrichment of serine via formation, dissociation, and soft-landing of octameric cluster ions.

Chiral enrichment of serine is achieved in experiments that involve formation of serine octamers starting from non-racemic serine solutions. Serine octamers were generated by means of electrospray and sonic spray ionization of aqueous solutions of d(3)-L-serine (108 Da) and D-serine (105 Da) having different molar ratios of enantiomers. A cyclic process involving the formation of chirally-enriched octameric cluster ions and their dissociation, viz. Ser(1) --> Ser(8) --> Ser(1), allows serine monomers to be regenerated with increased enantiomeric excess as shown in two types of experiments: (1) Chiral enrichment in serine was observed in MS/MS/MS experiments in a quadrupole ion trap in which the entire distribution of serine octamers formed from non-racemic solutions was isolated, collisionally activated, and fragmented. Monomeric serine was regenerated with increased enantiomeric excess upon dissociation of octamers when compared with the enantiomeric composition of the original solution. (2) Chiral enrichment was observed in the products of soft-landing of mass-selected protonated serine octamers. These ions were generated by means of electrospray or sonic spray ionization, mass selected, and collected on a gold surface using ion soft-landing. Chiral enrichment of the soft-landed serine was established by redissolving the recovered material and comparing the intensities of protonated molecular ions of d(3)-L-serine and D-serine after APCI-MS analysis. Both of these experiments showed comparable results, suggesting that formation of serine octamers depends only on the enantiomeric composition of the serine solution and that the magnitude of the chiral preference is intrinsic to octamers formed from solutions of given chiral composition.

Ions↗
PubMed2002

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.

Adult↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

Effects of serine mutations in transmembrane domain 7 of the human norepinephrine transporter on substrate binding and transport.

Two serine residues in the beta-adrenergic receptor (beta-AR) have been proposed to form hydrogen bonds with the catechol moiety of the ligand and contribute to the activation of the receptor. These conserved serine residues in the dopamine (DA) and norepinephrine transporters (DAT and NET, respectively) have also been shown to affect substrate transport in the rat DAT. In the present work, hydrogen bonding interactions between the corresponding serine residues in the human NET (hNET), 354 and 357, and the hydroxyl groups on the substrate were systematically evaluated by examining the transport and binding properties of DA and several single hydroxyl analogues of DA at wild-type and serine-to-alanine-substituted transporters. A comparison of [3H]nisoxetine binding at the serine 354 mutant, in which K(D) increased 70-fold from the wild-type value, with the binding of DA, m-tyramine (m-TYR), and p-tyramine (p-TYR) at mutant 354, where the increase in Ki was less dramatic, revealed that serine 354 is more influential in inhibitor than substrate binding. The binding of m-TYR and p-TYR at the serine 354 and serine 357 mutants did not show a direct interaction between one serine and one substrate catechol hydroxyl group. DA, m-TYR, and p-TYR binding affinity did not deviate from the wild-type value at the serine 357 and double mutant transporters. At these two transporters, however, the Km of DA uptake increased, suggesting that the roles of serine 357 and serine 354 in substrate transport are different from their roles in binding. The K'm for induced efflux of DA decreased at the serine 357 mutant compared with the wild-type, whereas the K'm at the serine 354 mutant was the same as that of the wild-type. Further investigation of the role of substrate hydroxyls in the transport process revealed no difference between the transport of m-TYR or p-TYR, as measured indirectly through their induced efflux of DA, at any of the mutants. Although these serines are influential in inhibitor and substrate binding to the transporter and substrate uptake and efflux, they do not appear to be involved in a direct hydrogen bond interaction with substrate, suggesting that the pattern of distinct hydrogen bonding interactions at the beta-AR does not exist at the hNET.

Animals↗
PubMed2004

Serine-deficiency syndromes.

PURPOSE OF REVIEW: Serine-deficiency disorders comprise a new group of neurometabolic diseases and are caused by defects in the biosynthesis of the amino acid L-serine. In contrast to most neurometabolic disorders, serine-deficiency disorders are potentially treatable. Furthermore, the severe neurological symptoms observed in patients underscore the important roles of the serine biosynthetic pathway in brain tissue. An overview of the patients with serine-deficiency disorders reported to date, the biochemical findings and the results of treatment with amino acids is presented. RECENT FINDINGS: L-Serine biosynthesis plays an important role in multiple cellular reactions, particularly in the brain, as L-serine is a precursor of important metabolites such as nucleotides, phospholipids and the neurotransmitters glycine and D-serine. Disturbances of serine-glycine metabolism in relation to N-methyl-D-aspartate-receptor activation are supposed to play a role in psychiatric disease as well. Recent findings concerning these roles of L-serine-derived phospholipids and neurotransmitters are presented. SUMMARY: Congenital microcephaly, seizures and severe psychomotor retardation are symptoms of serine deficiency and can be treated with supplementation of L-serine, sometimes combined with glycine. The symptoms observed in serine deficiency confirm that L-serine and L-serine-derived metabolites play important roles in the central nervous system.

Adult↗
PubMed2006

DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073.

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.

Alanine↗
PubMed1992

Fetal serine fluxes across fetal liver, hindlimb, and placenta in late gestation.

Eleven studies of fetal serine fluxes were performed in chronically catheterized fetal lambs by continuous infusion of [1-13C]- and [U-14C]serine into a fetal brachial vein. At tracer serine steady state, samples were collected from the fetal abdominal aorta, umbilical vein, fetal hepatic vein, and fetal femoral vein and from the maternal femoral artery and uterine vein. Analyses were performed for plasma serine and glycine concentration, for serine and glycine 13C mole percent enrichment, and for whole blood 14CO2 and O2 concentrations. Uterine and umbilical blood flows were also measured. The placenta had a significant net uptake of fetal serine (2.1 +/- 0.5 mumol.min-1.kg-1, P < 0.01). Fetal plasma serine disposal rate (DR) was 42.5 +/- 3.9 mumol.min-1.kg-1.CO2 production from decarboxylation of fetal plasma serine represented 7.9 +/- 0.5% of DR, or 10.1 +/- 1.2 mumol CO2.min-1.kg-1. Fetal plasma glycine enrichment was 59.7 +/- 4.9% of fetal plasma serine enrichment. There was a significant loss of tracer serine from the fetal circulation into the placenta accounting for approximately 45% of infused tracer. Fifteen percent of this was converted to glycine and released into the umbilical circulation. There was a significant uptake of tracer serine by both fetal liver and fetal hindlimb with a significant CO2 production by both sites with serine oxidation predominantly in the carcass. These results indicate a high fetal serine disposal rate in the lamb, with rapid fetoplacental serine exchange, resulting in a net uptake of fetal serine by the placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗