D-Amino acid oxidase of Streptomyces coelicolor and the effect of D-amino acids on the bacterium

The homologous gene of D-amino acid oxidase (DAO) in prokaryotic organisms is predominantly found in a group of bacteria called the Actinobacteria. We have analyzed the DAO of the model actinomycete Streptomyces coelicolor and the effect of D-amino acids on this bacterium. When expressed in Escherichia coli, the translated product of the putative dao gene of this bacterium exhibited oxidase activity against neutral and basic D-amino acids, with a higher activity toward D-valine and D-isoleucine, but not to their corresponding L-amino acids. This substrate specificity was largely different from that of the DAO of the actinobacterium Arthrobacter protophormiae. The gene message and DAO activity were constitutively detected in S. coelicolor cells, and unlike eukaryotic DAOs, the presence of a D-amino acid did not significantly induce expression. The D-amino acids that were a good substrate for S. coelicolor DAO inhibited cell growth, delayed morphological development and affected cell morphology, but they did not inhibit biofilm formation. Disruption of the dao gene had no effect on the morphology and morphological development of S. coelicolor cells, the assimilation of D-valine or the sensitivity to growth inhibition by D-valine under the experimental conditions, showing that in this bacterium DAO does not play a significant role in either morphological development or the assimilation and detoxification of D-amino acids.     

l-Amino acid oxidases from microbial sources: types,properties, functions,and applications

l-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of l-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotide-containing homodimeric proteins. l-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. Because expressing LAAOs as recombinant proteins in heterologous hosts is difficult, their biotechnological applications have not been thoroughly advanced. LAAOs are thought to contribute to amino acid catabolism, enhance iron acquisition, display antimicrobial activity, and catalyze keto acid production, among other roles. Here, we review the types, properties, structures, biological functions, heterologous expression, and applications of LAAOs obtained from microbial sources. We expect this review to increase interest in LAAO studies.     

Blood-brain barrier transport ofL-pipecolic acid in various rat brain regions

Blood-brain barrier transport ofL-[l-¹⁴C]pipecolic acid was studied in the rat by single intracarotid injection using³H₂O as a diffusible internal standard. Brain uptake index (BUI) forL-[¹⁴C]pipecolic acid (0.036 mM) was found to be 18.1, 10.5, and 12.6 for the cerebral cortex, brain stem, and cerebellum, respectively which was substantially higher than that reported for its analogL-proline in the whole brain. Influx ofL-pipecolic acid into the brain was concentration dependent and differed significantly between the cerebral cortex and the brain stem, and between the cerebral cortex and the cerebellum, but not between the brain stem and the cerebellum. Kinetic study ofL-pipecolic acid influx revealed a low- and a high-capacity uptake mechanisms. The low-capacity saturable component hasK _m values ranging from 38 to 73 μM, andV _max values ranging from 10 to 13 nmol/g/min for the three brain regions. The nonsaturable component has aK _m of 4 mM, aV _max of 200 nmol/g/min and similar diffusion constant (K _d) (0.03 to 0.06 mlg^?1 min^?1) for all three brain regions. A possible role of the two-component brain uptake mechanism in the regulation of the neuronal function ofL-pipecolic acid was suggested.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

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.     

Characterization and physiological function of a soluble l-amino acid oxidase in Corynebacterium

A general l-amino acid oxidase (l-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.2.) has been characterized in Corynebacterium. The enzyme is soluble (MW 130 000–140 000) and is active with most l-α-amino acids but not with aspartate, threonine, proline and glycine. It is subject to substrate inhibition. This amino acid oxidase is induced along with catalase by growth in the presence of amino acids as a nitrogen source and is repressed when ammonium ions are present in the medium. Its probable physiological function is to allow the utilization of amino acids as a nitrogen source.     

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.     

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-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.     

The role of d-amino acids in amyotrophic lateral sclerosis pathogenesis: a review

A potential role for d-amino acids in motor neuron disease/amyotrophic lateral sclerosis (ALS) is emerging. d-Serine, which is an activator/co-agonist at the N-methyl-d-aspartate glutamate receptor subtype, is elevated both in spinal cord from sporadic cases of ALS and in an animal model of ALS. Furthermore, we have shown that a mutation in d-amino acid oxidase (DAO), an enzyme strongly localized to spinal cord motor neurons and brain stem motor nuclei, is associated with familial ALS. DAO plays an important role in regulating levels of d-serine, and its function is impaired by the presence of this mutation and this may contribute to the pathogenic process in ALS. In sporadic ALS cases, elevated d-serine may arise from induction of serine racemase, its synthetic enzyme, caused by cell stress and inflammatory processes thought to contribute to disease progression. Both these abnormalities in d-serine metabolism lead to an increase in synaptic d-serine which may contribute to disease pathogenesis.     

Upstream molecular signaling pathways of p27(Kip1) expression in human breast cancer cells in vitro: differential effects of 4-hydroxytamoxifen and deficiency of either D-(+)-glucose or L-leucine

Background

The objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D-(+)-glucose could down-regulate and, conversely, (b) the deficiency of D-(+)-glucose or certain L-amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1.

Results

Using human MDA-MB-231 breast cancer cells in vitro, these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D-(+)-glucose or L-leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3.

Conclusions

(a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D-(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D-(+)-glucose or L-leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.     

New insights on the role of free d-aspartate in the mammalian brain

Free d-aspartate (d-Asp) occurs in substantial amounts in the brain at the embryonic phase and in the first few postnatal days, and strongly decreases in adulthood. Temporal reduction of d-Asp levels depends on the postnatal onset of d-aspartate oxidase (DDO) activity, the only enzyme able to selectively degrade this d-amino acid. Several results indicate that d-Asp binds and activates N-methyl-d-aspartate receptors (NMDARs). Accordingly, recent studies have demonstrated that deregulated, higher levels of d-Asp, in knockout mice for Ddo gene and in d-Asp-treated mice, modulate hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory. Moreover, similarly to d-serine, administration of d-Asp to old mice is able to rescue the physiological age-related decay of hippocampal LTP. In agreement with a neuromodulatory action of d-Asp on NMDARs, increased levels of this d-amino acid completely suppress long-term depression at corticostriatal synapses and attenuate the prepulse inhibition deficits produced in mice by the psychotomimetic drugs, amphetamine and MK-801. Based on the evidence which points to the ability of d-Asp to act as an endogenous agonist on NMDARs and considering the abundance of d-Asp during prenatal and early life, future studies will be crucial to address the effect of this molecule in the developmental processes of the brain controlled by the activation of NMDARs.     

Alteration of intrinsic amounts of d-serine in the mice lacking serine racemase and d-amino acid oxidase

For elucidation of the regulation mechanisms of intrinsic amounts of d-serine (d-Ser) which modulates the neuro-transmission of N-methyl-d-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and d-amino acid oxidase (DAO) were established, and the amounts of d-Ser in the tissues and physiological fluids were determined. d-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in d-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR^?/?) mice compared with those of control mice, although the amounts of d-Ser in these tissues were low. The amounts of d-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous d-Ser into the brain tissues, we have determined the d-Ser of SRR^?/? mice after oral administration of d-Ser for the first time, and a drastic increase in d-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic d-Ser amounts was investigated. In the frontal brain, most of the intrinsic d-Ser was biosynthesized by SRR. On the other hand, half of the d-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic d-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.     

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.     

D-amino acids in the cell pool of bacteria

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B₃,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B₃ varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O₂ supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.     

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.     

Cocaine kindling in mice

Previous studies proposed the involvement of theN-methyl-D-aspartate (NMDA) type of glutamate receptors in the development of sensitization to the convulsive effect of cocaine (cocaine kindling). The present study was undertaken to determine, first, if cocaine kindling is associated with enhanced sensitivity of the NMDA receptor to the convulsive response ofN-methyl-D,L-aspartate (NMDLA), and second, whether in vivo modulation of nitric oxide synthase (NOS) function regulates the development of cocaine kindling. The following results were observed:

1. Cocaine-kindled animals were significantly more susceptible to the convulsive effect of the NMDA receptor agonist NMDLA than saline controls;
2. Pretreatment with the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 100 mg/kg; ip) blocked the development of cocaine kindling;
3. The protective effect of L-NAME was partially reversed with the coadministration of the NOS substrate,L-arginine (300 mg/kg; ip), but notD-arginine; and
4. L-Arginine (300 mg/kg; ip), but notD-arginine, amplified the development of cocaine kindling. Taken together, these findings suggest that supersensitivity of the NMDA receptor and activation of NOS may underlie the development of cocaine kindling.

     

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-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.     

Amperometric microbiosensor as an alternative tool for investigation of d-serine in brain

This paper discusses the application of a reagentless, selective microbiosensor as a useful alternative tool for monitoring d-serine in neural samples. The main components of the 125-μm-diameter disk biosensor were d-amino acid oxidase for d-serine sensitivity (linear region slope, 61?±?7?μA?cm^–2?mM^–1; limit of detection, 20?nM), and poly-phenylenediamine for rejection of electroactive interference. The response time of the biosensor was of the order of 1?s, ideal for ‘real-time’ monitoring, and detection of systemically administered d-serine in brain extracellular fluid is demonstrated. Exploitation of this probe might resolve queries involving regulation of d-serine in excitotoxicity, and modulation of N-methyl-d-aspartate receptor function by d-serine and glycine in the central nervous system.