l-Theanine elicits umami taste via the T1R1 + T1R3 umami taste receptor

l-Theanine is a unique amino acid present in green tea. It elicits umami taste and has a considerable effect on tea taste and quality. We investigated l-theanine activity on the T1R1 + T1R3 umami taste receptor. l-Theanine activated T1R1 + T1R3-expressing cells and showed a synergistic response with inosine 5′-monophosphate. The site-directed mutagenesis analysis revealed that l-theanine binds to l-amino acid binding site in the Venus flytrap domain of T1R1. This study shows that l-theanine elicits an umami taste via T1R1 + T1R3.     

d-Amino acids in the brain and mutant rodents lacking d-amino-acid oxidase activity

d-Amino acids are stereoisomers of l-amino acids. They are often called unnatural amino acids, but several d-amino acids have been found in mammalian brains. Among them, d-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. d-Amino-acid oxidase (DAO), which degrades neutral and basic d-amino acids, is mainly present in the hindbrain. DAO catabolizes d-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of d-serine and other d-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of d-serine. d-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that d-amino acids and DAO have pivotal functions in the central nervous system.     

Serine racemase: an unconventional enzyme for an unconventional transmitter

The discovery of large amounts of d-serine in the brain challenged the dogma that only l-amino acids are relevant for eukaryotes. The levels of d-serine in the brain are higher than many l-amino acids and account for as much as one-third of l-serine levels. Several studies in the last decades have demonstrated a role of d-serine as an endogenous agonist of N-methyl-d-aspartate receptors (NMDARs). d-Serine is required for NMDAR activity during normal neurotransmission as well as NMDAR overactivation that takes place in neurodegenerative conditions. Still, there are many unanswered questions about d-serine neurobiology, including regulation of its synthesis, release and metabolism. Here, we review the mechanisms of d-serine synthesis by serine racemase and discuss the lessons we can learn from serine racemase knockout mice, focusing on the roles attributed to d-serine and its cellular origin.     

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and l-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of l-serine ammonia lyase was also examined. Specific activity was highest when l-serine levels in the hemolymph were highest. Thus, it appears that l-serine levels increased under hyposmotic conditions due to the consumption of l-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.     

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.     

Construction of an l-serine producing Escherichia coli via metabolic engineering

l-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, l-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce l-serine from glucose. To accumulate l-serine, sdaA encoding the l-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious l-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA ^FR , serB, and serC genes. The resulting strain YF-7 produced 4.5 g/L l-serine in batch cultivation and 8.34 g/L l-serine in fed-batch cultivation.     

Principal component analysis of the relationship between the d-amino acid concentrations and the taste of the sake

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1–7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with “Kimoto yeast starter” and “Yamahaimoto” had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with “Sokujo-moto”. When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50–5,000 μM dl-Ala did not effect on the aroma of the sakes at all.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

Catalytic mechanism of serine racemase from Dictyostelium discoideum

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Increasing effects of S-methyl-l-cysteine on the extracellular d-serine concentrations in the rat medial frontal cortex

In an in vivo dialysis experiment, the intra-medial frontal cortex infusion of a system A and Asc-1 transporter inhibitor, S-methyl-l-cysteine, caused a concentration-dependent increase in the dialysate contents of an endogenous coagonist for the N-methyl-d-aspartate (NMDA) type glutamate receptor, d-serine, in the cortical portion. These results suggest that these neutral amino acid transporters could control the extracellular d-serine signaling in the brain and be a target for the development of a novel threapy for neuropsychiatric disorders with an NMDA receptor dysfunction.     

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.     

Structure–function relationships in human d-amino acid oxidase

Since d-amino acids were identified in mammals, d-serine has been one of the most extensively studied “unnatural amino acids”. This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of d-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of d-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and d-amino acid oxidase (which catalyzes d-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human d-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of d-amino acid oxidase in brain d-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development.     

Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation

In this study, the d-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and d-serine has recently been demonstrated to be phytoinhibitory to plant growth. d-Serine ammonia lyase detoxifies d-serine via a substrate-specific reaction to pyruvate, ammonia, and water. d-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2?C15 mM. Transgenic plants were recovered in the presence of d-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM d-serine, indicating that d-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and non-transgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the d-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize d-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of d-serine represents a new and effective selectable marker system for maize transformation.     

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.     

d-Serine and Serine Racemase are Localized to Neurons in the Adult Mouse and Human Forebrain

d-Serine, a co-agonist at the NMDA receptor (NMDAR), is synthesized from l-serine by the enzyme serine racemase (SR), which is heavily expressed in the forebrain. Although SR was originally reported to be localized exclusively to astrocytes, recent conditional knock out results demonstrate that little SR is expressed in forebrain astrocytes. As a consequence, the cellular location of its product, d-serine, in the brain is also uncertain. Immunocytochemistry now indicates that SR is expressed primarily in forebrain glutamatergic neurons with the remainder in GABAergic interneurons. We utilized SR deficient (SR?/?) mice, which have <15 % of normal d-serine levels, to validate and optimize a d-serine immunohistochemical method. Nearly all of the d-serine in neocortex and hippocampus (HP) is found in neurons, with virtually no d-serine co-localizing with two astrocyte markers. Interestingly, only a subset of the d-serine positive neurons contained SR in the neocortex and HP. Greater than half of the d-serine positive neurons were GABAergic interneurons, with a majority of these neurons containing parvalbumin and/or somatostatin. Only ~25–40 % of interneurons expressed SR in the neocortex and HP. Finally, we demonstrate in human post-mortem neocortex that SR is found in both excitatory and inhibitory neurons, but not in S100β-containing astrocytes. In sum, these findings conclusively demonstrate that the majority of d-serine is both synthesized and stored in neurons. It will be important to determine the functional significance for the separation of synthesis and storage of d-serine in neurons, as well as the presence of this NMDAR co-agonist in GABAergic interneurons.     

Synthesis of enantiomeric pureCis-dichloroplatinum(II) amino acid complexes

The reaction of potassium tetrachloroplatinate(II) with six representative sulfurcontaining amino acids, namely,d- andl-cysteine,d- andl-methionine and its methyl ester hydrochloride gives the corresponding enantiomerically purecis-dichloroplatinum(II) complexes. This represents the first reported series of well-characterized enantiomerically pure platinum(II) complexes for bothd- andl-amino acids. The spectroscopic properties, including IR,¹H-NMR, and¹³C NMR, of these complexes and their configuration are discussed.     

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.     

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.