An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.     

Aromatic amino acid catabolism in trypanosomatids

Trypanosomatids cause important human diseases, like sleeping sickness, Chagas disease, and the leishmaniases. Unlike in the mammalian host, the metabolism of aromatic amino acids is a very simple pathway in these parasites. Trypanosoma brucei and Trypanosoma cruzi transaminate the three aromatic amino acids, the resulting 2-oxo acids being reduced to the corresponding lactate derivatives and excreted. In T. cruzi, two enzymes are involved in this process: a tyrosine aminotransferase (TAT), which despite a high sequence similarity with the mammalian enzyme, has a different substrate specificity; and an aromatic L-2-hydroxyacid dehydrogenase (AHADH), which belongs to the subfamily of the cytosolic malate dehydrogenases (MDHs), yet has no MDH activity. In T. cruzi AHADH the substitution of Ala102 for Arg enables AHADH to reduce oxaloacetate. In the members of the 2-hydroxyacid dehydrogenases family, the residue at this position is known to be responsible for substrate specificity. T. cruzi does not possess a cytosolic MDH but contains a mitochondrial and a glycosomal MDH; by contrast T. brucei and Leishmania spp. possess a cytosolic MDH in addition to glycosomal and mitochondrial isozymes. Although Leishmania mexicana also transaminates aromatic amino acids through a broad specificity aminotransferase, the latter presents low sequence similarity with TATs, and this parasite does not seem to have an enzyme equivalent to T. cruzi AHADH. Therefore, these closely related primitive eukaryotes have developed aromatic amino acid catabolism systems using different enzymes and probably for different metabolic purposes.     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl -dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.     

Sensitization to p-amino aromatic compounds: Study of the covalent binding of 2,5-dimethyl-p-benzoquinonediimine to a model peptide by electrospray ionization tandem mass spectrometry

To understand the hapten-protein complex formation in the context of skin contact allergy to p-amino aromatic derivatives, 2,5-dimethyl-p-benzoquinonediimine was used as a model compound to study the reactivity of p-benzoquinonediimines, first oxidation intermediates of allergenic p-amino aromatic compounds, toward a model peptide containing naturally occurring and potential reactive amino acids. LC-MS analysis, together with electrospray ionization MS/MS, was used for the determination of amino acid selectivity by studying the chemical modifications induced on the peptide due to covalent binding of the p-benzoquinonediimine. Results reported in this paper indicated that 2,5-dimethyl-p-benzoquinonediimine reacted with the epsilon-NH(2) group of lysine to first form a covalent adduct of the Schiff's base kind. Besides, an oxido-reduction process started that induced an oxidative deamination of lysine to form a peptidyl alpha-aminoadipic-delta-semialdehyde, by a mechanism similar to the one known for several enzymatic quinonoid co-factors, followed by an intramolecular cyclization of the peptide. From these results it could be concluded that lysine must be considered as an important amino acid for the hapten-protein complex formation in the case of p-benzoquinonediimines and that, in addition to direct covalent binding, further degradation of the peptide can be produced.     

Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen.

Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium.     

Amyloid‐like aggregation of designer bolaamphiphilic peptides: Effect of hydrophobic section and hydrophilic heads

Amyloid‐like aggregation of natural proteins or polypeptides is an important process involved in many human diseases as well as some normal biological functions. Plenty of works have been done on this ubiquitous phenomenon, but the molecular mechanism of amyloid‐like aggregation has not been fully understood yet. In this study, we showed that a series of designer bolaamphiphilic peptides could undergo amyloid‐like aggregation even though they didn't possess typical β‐sheet secondary structure. Through systematic amino acid substitution, we found that for the self‐assembling ability, the number and species of amino acid in hydrophobic section could be variable as long as enough hydrophobic interaction is provided, while different polar amino acids as the hydrophilic heads could change the self‐assembling nanostructures with their aggregating behaviors affected by pH value change. Based on these results, novel self‐assembling models and aggregating mechanisms were proposed, which might provide new insight into the molecular basis of amyloid‐like aggregation.     

Identification of peptides containing tryptophan, tyrosine, and phenylalanine using photodiode-array spectrophotometry

The characteristic absorption spectra of aromatic amino acids between 240 and 310 nm were used to identify tryptophan, tyrosine, and phenylalanine-containing peptides. In acidic solution, the absorption spectra of these amino acids exhibit minima or maxima at 255, 270, and 286 nm. Based on these characteristics, the content of the aromatic amino acid in peptide can be estimated. For this study, 2 nmol of tryptic peptides from human apolipoprotein A-1 was separated by high-performance liquid chromatography using a reverse-phase column. The peptide fragments were monitored by a photodiode-array spectrophotometer. This new approach offers a rapid, simple, sensitive, and direct identification of peptides containing aromatic amino acids. Those containing Trp, which may be of interest for DNA sequencing and important in sequence analysis of proteins, can be selectively purified using this technique.     

II. Excitotoxic models for neurodegenerative disorders

In recent years, considerable interest has been shown in the neurotoxic properties of excitatory amino acids and their possible relevance for the study of human neurodegenerative disorders. The term “excitotoxin” has been coined for a family of acidic amino acids which are neuroexcitants and produce a characteristic type of “axon-sparing” neuronal lesion. Intracerebral infusions of kainic and ibotenic acids, the two most commonly used excitotoxins, result in a morphological and biochemical picture in experimental animals which resembles that observed in the brains of Huntington's disease and epilepsy victims. The emergence of such animal models for neurodegenerative disorders has led to the hypothesis that endogenous excitotoxins may exist which are linked to the pathogenesis of human diseases. The most promising candidate discovered so far is quinolinic acid, a hepatic tryptophan metabolite which has recently also been found to occur in brain tissue. The particular excitotoxic properties of quinolinic acid warrant a thorough investigation of its metabolic and synaptic disposition in normal and abnormal brain function. While little is known about the mechanisms by which excitotoxins cause selective neuronal death, most current speculations propose the participation of specific synaptic receptors for acidic amino acids. The recent development of selective antagonists of such receptors has aided in the elucidation of excitotoxic mechanisms. Although a biochemical link between endogenous excitotoxins and human neurodegenerative disorders remains elusive at present, pharmacological blockade of excitotoxicity may constitute a novel therapeutic strategy for the treatment of these disease states.     

大肠杆菌高效合成L-苯甘氨酸的研究进展

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域.其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素Ⅰ等β-内酰胺类抗生素的生物合成当中.目前L苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不...     

The nitration of proteins in platelets

Nitric oxide has many important physiological functions, but it may also form an important oxidant, peroxynitrite, as a consequence of its reaction with superoxide anions. Peroxynitrite is capable of nitrating the aromatic amino acids in proteins, particularly tyrosine. Nitrated proteins are found in tissues of a variety of diseases where inflammation occurs. However, our recent work suggests that more selective nitration of specific proteins may occur during normal physiological processes, such as platelet activation by collagen. It is not yet clear what role this may play in the normal cell biology, but there is potential to be a role in signal transduction mechanisms, possibly by influencing tyrosine phosphorylation or dephosphorylation.     

Root exudates of plants

Summary The composition of the root exudates of barley and wheat in the initial growth phases was investigated; amino acids, organic acids, sugars and certain aromatic compounds could be identified. A knowledge of the composition of root exudates is important from the standpoint of the interaction between the plants and the micro-organisms in the rhizosphere. Some aspects of the rhizosphere effect connected with the present work are discussed.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review

The quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.

     

Radical formation in single crystals of aromatic amines and radical transformation by light

Single crystals of the four aromatic bioamine salts phenylethylamine hydrochloride, tyramine hydrochloride, tryptamine hydrochloride, and histamine dihydrochloride were grown in various states of deuteration. Free radicals were produced by exposure to X-rays between 77 and 300 K and investigated by electron spin resonance spectroscopy. Dissociation of atomic hydrogen from C beta of the aliphatic chain occurs in all compounds studied except tryptamine. However deamination as usually present in the analogous amino acids is not found. The C beta-radical is characterized by an anisotropic H alpha-splitting and two isotropic H beta-splittings. The latter splittings depend strongly on temperature in tyramine. In comparison to the analogous amino acids, radical formation in the aromatic residues is favoured. Among the seven different aromatic radicals found only one is identified in histamine but two in each of the three other bioamines. Two of these are characterized by hydrogen dissociation which occurs in phenylethylamine and tyramine. One hydrogen addition radical is found in each of the three compounds phenylethylamine, tyramine and histamine. In tryptamine two different addition radicals are detected. One of the two products can be converted into the other by visible light. The reverse process is induced by heat, thus permitting the switching of the radical site reversibly between two different structures.     

Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters

A cDNA was isolated from rat small intestine by expression cloning which encodes a novel Na+-independent transporter for aromatic amino acids. When expressed in Xenopus oocytes, the encoded protein designated as TAT1 (T-type amino acid transporter 1) exhibited Na+-independent and low-affinity transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine (Km values: approximately 5 mm), consistent with the properties of classical amino acid transport system T. TAT1 accepted some variations of aromatic side chains because it interacted with amino acid-related compounds such as l-DOPA and 3-O-methyl-DOPA. Because TAT1 accepted N-methyl- and N-acetyl-derivatives of aromatic amino acids but did not accept their methylesters, it is proposed that TAT1 recognizes amino acid substrates as anions. Consistent with this, TAT1 exhibited sequence similarity (approximately 30% identity at the amino acid level) to H+/monocarboxylate transporters. Distinct from H+/monocarboxylate transporters, however, TAT1 was not coupled with the H+ transport but it mediated an electroneutral facilitated diffusion. TAT1 mRNA was strongly expressed in intestine, placenta, and liver. In rat small intestine TAT1 immunoreactivity was detected in the basolateral membrane of the epithelial cells suggesting its role in the transepithelial transport of aromatic amino acids. The identification of the amino acid transporter with distinct structural and functional characteristics will not only facilitate the expansion of amino acid transporter families but also provide new insights into the mechanisms of substrate recognition of organic solute transporters.     

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.     

Amino acids modulate calcium permeability of the plasma membrane of human neuroblastoma cells

Four different amino acids (kainate, N-methyl-D-aspartate, L-cysteine sulfinate and D,L-2-amino-5-phosphonovalerate) have been observed to stimulate uptake of 45Ca2+ into human neuroblastoma cells. This stimulation of uptake is specific and many amino acids which are structural analogs of the above compounds are without activity. The calcium movement is not inhibited by compounds which block voltage-dependent calcium channels. Biological specificity is observed in which some cell lines respond to the amino acids and others do not. It is concluded that these amino acids are acting on a class of receptors whose physiological role is modulation of neuronal metabolism by modulating the calcium permeability of the plasma membrane. The amino acids can substitute for the, as yet, unidentified natural agonists, albeit with low affinity.     

Aromatic Amino Acid Activation of Signaling Pathways in Bone Marrow Mesenchymal Stem Cells Depends on Oxygen Tension

The physiologic oxygen pressures inside the bone marrow environment are much lower than what is present in the peripheral circulation, ranging from 1–7%, compared to values as high as 10–13% in the arteries, lungs and liver. Thus, experiments done with bone marrow mesenchymal stem cells (BMMSCs) using standard culture conditions may not accurately reflect the true hypoxic bone marrow microenvironment. However, since aging is associated with an increased generation of reactive oxygen species, experiments done under 21%O₂ conditions may actually more closely resemble that of the aging bone marrow environment. Aromatic amino acids are known to be natural anti-oxidants. We have previously reported that aromatic amino acids are potent agonists for stimulating increases in intracellular calcium and phospho-c-Raf and in promoting BMMSC differentiation down the osteogenic pathway. Our previous experiments were performed under normoxic conditions. Thus, we next decided to compare a normoxic (21% O₂) vs. a hypoxic environment (3% O₂) alone or after treatment with aromatic amino acids. Reverse-phase protein arrays showed that 3% O₂ itself up-regulated proliferative pathways. Aromatic amino acids had no additional effect on signaling pathways under these conditions. However, under 21%O₂ conditions, aromatic amino acids could now significantly increase these proliferative pathways over this “normoxic” baseline. Pharmacologic studies are consistent with the aromatic amino acids activating the extracellular calcium-sensing receptor. The effects of aromatic amino acids on BMMSC function in the 21% O2 environment is consistent with a potential role for these amino acids in an aging environment as functional anti oxidants.     

Malate metabolism by NADP-malic enzyme in plant defense

Malate is involved in various metabolic pathways, and there are several enzymes that metabolize it. One important malate metabolizing enzyme is NADP-malic enzyme (NADP-ME). NADP-ME functions in many different pathways in plants, having an important role in C₄ photosynthesis where it releases the CO₂ to be used in carbon fixation by Rubisco. Apart from this specialized role, NADP-ME is thought to fulfill diverse housekeeping functions because of its universal presence in different plant tissues. NADP-ME is induced after wounding or exposure to UV-B radiation. In this way, the enzyme is implicated in defense-related deposition of lignin by providing NADPH for the two NADPH-dependent reductive steps in monolignol biosynthesis. On the other hand, it can supply NADPH for flavonoid biosynthesis as many steps in the flavonoid biosynthesis pathway require reductive power. Pyruvate, another product of NADP-ME reaction, can be used for obtaining ATP through respiration in the mitochondria; and may serve as a precursor for synthesis of phosphoenolpyruvate (PEP). PEP is utilized in the shikimate pathway, leading to the synthesis of aromatic amino acids including phenylalanine, the common substrate for lignin and flavonoid synthesis. Moreover, NADP-ME can be involved in mechanisms producing NADPH for synthesis of activated oxygen species that are produced in order to kill or damage pathogens. In conclusion, an increase in the levels of NADP-ME could provide building blocks and energy for biosynthesis of defense compounds, suggesting a role of malate metabolism in plant defense.