Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus

The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000 but were distinguished by their different affinities to the two substrates, l-threonine and l-serine.Abbreviations SDH l-serine dehydratase - TDH l-threonine dehydratase     

Entrapment of Lavandula vera cells with synthetic resin prepolymers and its application to pigment production

Summary Cultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.Dedicated to Professor Dr. Georg Manecke on occasion of his 70th birthday     

Metabolic engineering and flux analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for L-serine production

l-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of l-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of l-serine to pyruvate and glycine, releasing the feedback inhibition by l-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH^r). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in l-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular l-serine pool reached (14.22±1.41) μmol g_CDM ⁻¹, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH^r improved the l-serine biosynthesis pathway. In addition, the flux from l-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that l-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C₁ units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for l-serine production in C. glutamicum.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

Immobilization of <Emphasis Type="Italic">Streptomyces</Emphasis> phospholipase D on a Dowex macroporous resin

The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme, which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%) was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l -serine was employed again after replacing the amount that was used.     

Ionic dependence of amino-acid transport in the exocrine pancreatic epithelium: Calcium dependence of insulin action

Summary Rapid unidirectional transport (15 sec) ofl-serine and 2-methylaminoisobutyric acid (MeAIB) was studied in the isolated perfused rat pancreas using a dual-tracer dilution technique. Time-course experiments in the presence of normal cation gradients revealed a time-dependent transstimulation ofl-serine influx and transinhibition of MeAIB influx. Transport of the model nonmetabolized System A analog MeAIB was Na⁺ dependent and significantly inhibited during perfusion with 1mm ouabain. Although transport ofl-serine was largely Na⁺ independent, ouabain caused a time-dependent inhibition of transport. Influx of both amino acids appeared to be inhibited by the ionophore monensin but unaffected by a lowered extracellular potassium concentration. Removal of extracellular calcium had no effect on influx of the natural substratel-serine, whereas stimulation of transport by exogenous insulin (100 U/ml) was entirely dependent upon extracellular calcium and unaffected by ouabain. Paradoxically, exogenous insulin had no effect on the time-course of MeAIB influx. The characteristics ofl-serine influx described in earlier studies together with our present findings suggest that insulin may modulate the activity of System asc in the exocrine pancreatic epithelium by a calcium-dependent mechanism.     

Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Spectroscopic,theoretical and structural characterization of hydrogensquarates of <Emphasis Type="SmallCaps">l</Emphasis>-threonyl-<Emphasis Type="SmallCaps">l</Emphasis>-serine and <Emphasis Type="SmallCaps">l</Emphasis>-serine

Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, ¹H- and ¹³C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical predicted structures and experimentally obtained by single crystal X-ray diffraction.     

Synthesis of l-dopa by escherichia intermedia cells immobilized in a polyacrylamide gel

Summary Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l^-1 l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l^-1·h^-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation.     

Characterization of thermophilicCampylobacter: I. Carbon-substrate utilization tests

The carbon-substrate utlization profile of 234 wild strains of thermophilic campylobacters originating from different animal sources and different part of the world was studied using a microgallery as well as the profile of 25 type strains ofCampylobacter species and reference strains ofCampylobacter-like organisms. Among the 98 substrates tested, succinate, fumarate,d-l-lactate,l-malate, pyruvate,l-glutamate,l-aspartate, andl-serine (with one exception for the last two) were always utilized by the wild strains, and acetate, propionate,d-malate, 2-cetoglutarate, itaconate, citrate, andl-proline by some of the strains. A strong association was found between assimilation ofd-malate and a positive hippurate test.     

Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus)

Summary Intracellular calcium was measured in single olfactory neurons from the channel catfish (Icatalurus punctatus) using the fluorescent Ca²⁺ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (l-alanine,l-arginine,l-norleucine andl-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding ofl-alanine andl-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction.     

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

Histochemical demonstration of l-amino acid-tetrazolium reductase

Summary A method for the histochemical demonstration of l-amino acid-tetrazolium reductase is described. The diformazan deposition was obtained with l-leucine as substrate and no precipitate was present when l-serine and l-lisine were used. In the kidney the reaction was positive in the podocytes, the cells of proximal and distal convoluted tubules, in the ascending limbs of Henle and in the cells of the collecting tubules. In the liver the reaction was positive in the cytoplasm of the hepatocytes. The reaction pattern suggests that it is predominantly extramitochondrial. The specificity of this method is discussed.     

Expression of <Emphasis Type="SmallCaps">l</Emphasis>-Serine Biosynthetic Enzyme 3-Phosphoglycerate Dehydrogenase (Phgdh) and Neutral Amino Acid Transporter ASCT1 Following an Excitotoxic Lesion in the Mouse Hippocampus

The nonessential amino acid l-serine functions as a glia-derived trophic factor and strongly promotes the survival and differentiation of cultured neurons. The l-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (Phgdh) and the small neutral amino acid transporter ASCT1 are preferentially expressed in specific glial cells in the brain. However, their roles in pathological progression remain unclear. We examined the expression of Phgdh and ASCT1 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus using immunohistochemistry and Western blots. Our quantitative analysis revealed that Phgdh and ASCT1 were constitutively expressed in the normal brain and transiently upregulated by KA-treatment. At the cellular level, Phgdh was expressed in astrocytes in control and in KA-treated mice while ASCT1 that was expressed primarily in the neurons of the normal brain appeared also in activated astrocytes in KA treated mouse brain. The preferential glial expression of ASCT1 was consistent with that of Phgdh. These results demonstrate injury-induced changes in Phgdh and ASCT1 expression. It is hypothesized that the secretion of l-serine is regulated by astrocytes in response to toxic molecules such as glutamate and free radicals that promote neurodegeneration, and may correspond to the level of l-serine needed for neuronal survival and glial activation during brain insults. G. S. Jeon and D. H. Choi contributed equally to this work.     

Investigation of the origin of 0-methyl groups in griseofulvin using some14C-labelled substrates

I-^l4C-pyruvie acid, 3-¹⁴C-l-serine,¹⁴C-formic acid and¹⁴CO₂ were tested as possible sources of 0-methyl groups of griseofulvin produced byPenicillium griseofulvum. Entire radioactivity from pyruvic acid,l-serine and formic acid was found in the methoxyls of griseofulvin. By determining the activity of individual methoxyls its distribution was established, this being homogeneous only after formic acid.     

Facile syntheses of <Emphasis Type="SmallCaps">l</Emphasis>-β-haloalanine derivatives from <Emphasis Type="SmallCaps">l</Emphasis>-cysteine or <Emphasis Type="SmallCaps">l</Emphasis>-cystine

l-β-Haloalanines are physiologically active unnatural amino acids and they are useful intermediates for the synthesis of natural and unnatural amino acids, S-linked glycopeptides, and lanthionines. In general l-β-haloalanines were prepared predominantly from l-serine via functional group transformation. Here we reported an alternative approach for the preparation of l-β-haloalanines via halogenation of protected l-cysteine esters which was obtained from l-cysteine or l-cystine, respectively. The mercapto group of protected l-cysteine esters was efficiently transformed to halo groups by triphenylphosphine/N-halosuccinimides. It has been proved to be a versatile desulfurization strategy via this functional group transformation.     

Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources,antibiotics and metabolic inhibitors

The effect of nitrogen and carbon sources, vitamins, antibiotics and metabolic inhibitors on growth and differentiation ofPaecilomyces viridis was investigated. Sodium nitrate,l-asparagine,l-proline and peptone were found to be suitable nitrogen sources for mycelial growth (M) in a synthetic medium with glucose.Paecilomyces viridis could also grow slowly in a synthetic medium containing benzylpenicillin or bacitracin as the only nitrogen sources and very slowly even in a medium with polymyxin as the nitrogen source. Ammonium salts, area,l-arginine,d, l-aspartic acid andl,-serine were found to support intensive sporulation. Partially yeast-like growth (Y) was facilitated by NaNO₂, (NH₄)₂SO₄, NH₄NO₃, urea,d, l-alanine,l-arginine,d, l-aspartic acid,l-cysteine,l-glutamic acid andl-serine. Partially yeastlike growth could be observed in a medium with peptone and at an initial pH of 2. The following compounds appear as suitable carbon sources for mycelial growth:d-glucose,d-galactose,d-mannose, maltose, sucrose, chitin andd-mannitol. No changes in morphology could be detected on any of the 25 used carbon sources in a synthetic medium with NaNO₃. Yeast-like growth was induced by the antibiotics azalomycin F, cyanein (brefeldin A), griseofulvin and monorden (radicicol). After removal of the antibiotics, mycelial growth was restored. Sporulation was stimulated by chloramphenicol, 2-deoxy-d-glucose, furancarboxylic acid and stipitatic acid. Deformation of phialides was observed after treatment with actinomycin D, amphotericin B, boromycin, citrinin, cycloheximide, cytochalasin D, fungicidin and scopathricin. Microcyclic conidiation or growth of phialides directly from conidia were induced by cycloheximide, desertomycin, ethidium bromide and 5-fluorouracil.     

Localization and expression of serine racemase in Arabidopsis thaliana

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.