Enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different Corynebacterium glutamicum

During l-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (PCx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. To explore the significance of PCx for l-glutamate overproduction, the pyc gene encoding PCx was amplified in Corynebacterium glutamicum GDK-9 triggered by biotin limitation and CN1021 triggered by a temperature shock, respectively. In the fed-batch cultures, GDK-9pXMJ19pyc exhibited 7.4 % lower l-alanine excretion and no improved l-glutamate production. In contrast, CN1021pXMJ19pyc finally exhibited 13 % lower l-alanine excretion and identical l-glutamate production, however, 8.5 % higher l-glutamate production was detected during a short period of the fermentation. It was indicated that pyc overexpression in l-glutamate producer strains, especially CN1021, increased the supply of oxaloacetate for l-glutamate synthesis and decreased byproduct excretion at the pyruvate node.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Functional cardiovascular action of l-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat

The endogenous sulfur-containing amino acid l-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to l-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to l-glutamate (10 mM, 34 nl), microinjections of l-cysteine increased ABP and HR dose dependently (3–100 mM, 34 nl). The cardiovascular responses to l-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to l-cysteine. The results indicate that l-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to l-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of l-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.     

Neuroprotective Effects of Biochanin A Against Glutamate-Induced Cytotoxicity in PC12 Cells Via Apoptosis Inhibition

l-Glutamate plays a crucial role in neuronal cell death, which is known to be associated with various neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this study, we investigated the protective effects of biochanin A, a phytoestrogen compound found mainly in Trifolium pratense, against l-glutamate-induced cytotoxicity in a PC12 cell line. Exposure of the cells to 10 mM l-glutamate was found to significantly increase cell viability loss and apoptosis, whereas pretreatment with various concentrations of biochanin A attenuated the cytotoxic effects of l-glutamate. Specifically, the pretreatment led to not only decreases in the release of lactate dehydrogenase, the number of apoptotic cells, and the activity of caspase-3 but also an increase in the total glutathione level in the l-glutamate-treated PC12 cells. These results indicate that biochanin A may be able to exert neuroprotective effects against l-glutamate-induced cytotoxicity. Furthermore, our findings also imply that biochanin A may act as an antiapoptotic agent in order to perform its protective function.     

The Synthesis of l-dopa-l-Tyr and The Interaction of l-dopa-l-Tyr With ctDNA

l-dopa-l-Tyr was synthesized by Fmoc solid-phase peptide synthesis, purified by reversed-phase HPLC and characterized by using ¹H, ¹³C NMR and ESI–MS analyses. The interaction of l-dopa-l-Tyr and l-dopa with ctDNA has been investigated respectively by UV–vis absorption and fluorescence spectroscopy. The results showed that both l-dopa and l-dopa-l-Tyr interacted with ctDNA through intercalative mode and l-dopa-l-Tyr showed a higher affinity for DNA. Meanwhile, compared with the free l-dopa, gel electrophoresis assay also demonstrated that l-dopa-l-Tyr interacted with DNA by intercalation.     

Glutamate transport and xanthan gum production in the plant pathogen Xanthomonas axonopodis pv. citri

l-glutamate plays a central role in nitrogen metabolism in all living organisms. In the genus Xanthomonas, the nitrogen nutrition is an important factor involved in the xanthan gum production, an important exopolysaccharide with various industrial and biotechnological applications. In this report, we demonstrate that the use of l-glutamate by the phytopathogen Xanthomonas axonopodis pv. citri as a nitrogen source in defined medium significantly increases the production of xanthan gum. This increase is dependent on the l-glutamate concentration. In addition, we have also characterized a glutamate transport system that is dependent on a proton gradient and on ATP and is modulated by amino acids that are structurally related to glutamate. This is the first biochemical characterization of an energy substrate transport system observed in a bacterial phytopathogen with a broad economic and industrial impact due to xanthan gum production.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l-γ-glutamylamines producing 5-oxo-l-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l-γ-glutamylamines. The isodipeptide N ^?-(l-γ-glutamyl)-l-lysine 1 was used as a reference. The kinetic constants of the l-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l-γ-glutamyl amino acids except for l-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l-γ-glutamylamines restored activity for gGACT, and l-γ-glutamylneohexylamine 19 had a higher specificity constant (k _cat /K _m) than 1. gGACT did not exhibit any stereospecificity in the amide region of l-γ-glutamylamine substrates. In addition, analogues (26–30) with heteroatom substitutions for the γ methylene position of the l-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l-cysteine (28–30) were excellent substrates for gGACT.     

Development of a two-stage feeding strategy based on the kind and level of feeding nutrients for improving fed-batch production of l-threonine by Escherichia coli

Fed-batch fermentation is the predominant method for industrial production of amino acids. In this study, we comprehensively investigated the effects of four kinds of feeding nutrients and developed an accurate optimization strategy for fed-batch production of l-threonine. The production of l-threonine was severely inhibited when cell growth ceased in the bath culture. Similarly, l-threonine production was also associated with cell growth in the carbon-, phosphate-, and sulfate-limited fed-batch cultures, but the accumulation of l-threonine was markedly increased because of the extended production time in the growth stage. Interestingly, auxotrophic amino acid (l-isoleucine)-limited feeding promoted l-threonine production over the non-growth phase. Metabolite analysis indicates that substantial production of acetate and glutamate and the resulting accumulation of ammonium may lead to the inhibition of l-threonine production. During the growth phase, the levels of l-isoleucine were accurately optimized by balancing cell growth and production with Pontryagin’s maximum principle, basing on the relationship between the specific growth rate μ and specific production rate ρ. Furthermore, the depletion of l-isoleucine and phosphate at the end of the growth phase favored the synthesis of l-threonine in the subsequent non-growth phase. Combining the two-stage feeding profiles, the final l-threonine concentration and conversion rate were increased by 5.9- and 2.1-fold, respectively, compared to batch processes without feeding control. The identification of efficient feeding nutrient and the development of accurate feeding strategies provide potential guidelines for microbial production of amino acids.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Enantiomer-specific selection of amino acids

Dietary intake of l-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: (1) during long-, but not short-, term preference tests, l-alanine and l-serine were preferred over their d-enantiomer counterparts, while no such effect was observed for l-threonine vs. d-threonine; (2) these behavioral patterns were closely associated with the ability of l-amino acids to promote increases in respiratory exchange ratios such that those, and only those, l-amino acids able to promote increases in respiratory exchange ratios were preferred over their d-isomers; (3) these behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids and suggest a mechanistic link between substrate utilization and amino acid preferences.     

Properties, metabolisms, and applications of l-proline analogues

Due to the unique role of l-proline in the folding and structure of protein, a variety of synthetic proline analogues have been developed. l-Proline analogues have been proven to be valuable reagents for studying cellular metabolism and the regulation of macromolecule synthesis in both prokaryotic and eukaryotic cells. In addition to these fundamental researches, they are useful compounds for industrial use. For instance, microorganisms that overproduce l-proline have been obtained by isolating mutants resistant to l-proline analogues. They are also promising candidates for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring or de novo designed peptides. Among l-proline analogues, l-azetidine-2-carboxylic acid (l-AZC) is a toxic non-proteinogenic amino acid originally found in lily of the valley plants and trans-4-hydroxy-l-proline (4-l-THOP) is the most abundant component of mammalian collagen. Many hydroxyprolines (HOPs), such as 4-l-THOP and cis-4-hydroxy-l-proline (4-l-CHOP), are useful chiral building blocks for the organic synthesis of pharmaceuticals. In addition, l-AZC and 4-l-CHOP, which are potent inhibitors of cell growth, have been tested for their antitumor activity in tissue culture and in vivo. In this review, we describe the recent discoveries regarding the physiological properties and microbial production and metabolism of l-proline analogues, particularly l-AZC and HOPs. Their applications in fundamental research and industrial use are also discussed.     

d-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems

d-Aspartate (d-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of d-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of d-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by d-Asp application. d-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that d-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter—that the molecule’s biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for d-Asp’s biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although d-Asp receptors remain to be characterized, the postsynaptic response of d-Asp has been studied and several l-glutamate receptors are known to respond to d-Asp. In this review, we discuss the current status of research on d-Asp in neuronal and neuroendocrine systems, and highlight results that support d-Asp’s role as a signaling molecule.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

l-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of l-leucine and l-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of l-methionine and l-ethionine in the same manner as previously described l-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.     

Arginine increases genotoxicity induced by methyl methanesulfonate in human lymphocytes

Nitric oxide (NO) is a free radical that is produced in cells from l-arginine. NO is involved in the physiological control of different tissues, but it can act as a toxic mediator in the cells. In this study we investigated the effect of l-arginine on the genotoxicity induced by methyl methanesulfonate (MMS) in human lymphocytes. Blood was treated with N^G-nitro-l-arginine methyl ester (l-NAME) as an inhibitor of nitric oxide synthase for finding out the role of NO in this effect. Human whole blood was treated with l-arginine (50, 100 and 250 μM) and/or l-NAME, then it was treated in vitro with MMS after 24 h of culture. The lymphocytes were stimulated by phytohemagglutinin to find out the micronuclei in cytokinesis-blocked binucleated cells. DNA fragmentation of lymphocytes was detected by using a fluorescence microscope after propidium iodide staining. These data showed that arginine increased the frequency of MMS-induced micronuclei in lymphocytes. However, the genotoxicity was decreased by using l-NAME. Arginine and l-NAME have not shown any DNA damage in cultured human lymphocytes. In conclusion, addition of l-arginine to MMS as an alkylating agent caused an increase of DNA damage in human lymphocytes. This enhancement of genotoxicity was reduced by NAME as NO inhibitor. It is thus cleared that an increase of DNA damage by arginine and MMS is related to NO production.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.