Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Rickettsial pathogen uses arthropod tryptophan pathway metabolites to evade reactive oxygen species in tick cells

Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l ‐tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l ‐kynurenine levels that subsequently affects build‐up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l ‐kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3‐hydroxyl l ‐kynurenine limits A. phagocytophilum growth in tick cells. RNAi‐mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l ‐tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Effect of high tyrosine content on the determination of tryptophan in protein by the acidic ninhydrin method. Application to chicken ovoinhibitor

Colorimetric determination of tryptophan in intact proteins by the acidic ninhydrin method of Gaitonde & Dovey (1970) gives high apparent tryptophan contents for proteins having high tyrosine/tryptophan ratios. Correction for this interference by tyrosine can be achieved by plotting the ratio of observed to expected tryptophan content as a function of tyrosine/tryptophan ratio for proteins of known composition. The equation of the line is: [Formula: see text] Application of this correction to chicken ovoinhibitor, which contains 17 tyrosine residues per molecule, gave results that agree with tryptophan content determined by other methods.     

Effect of testosterone and 6-hydroxydopamine treatment on the metabolism of catecholamine and 5-hydroxytryptamine in methylcholanthrene-induced prostate carcinoma of rats.

The precursors tyrosine and tryptophan as well as the synthesizing and deaminating enzymes of catecholamines have been identified in methylcholanthrene-induced prostatic carcinoma of rats. Tyrosine hydroxylase, monoamine oxidase, catechol O-methyltransferase, dopamine, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid seemed to be neoplastic in origin, since electron microscopic studies failed to reveal the presence of any neuronal elements in this squamous epithelial cell carcinoma. Castration of rats significantly reduced the activity of tyrosine hydroxylase and the levels of tyrosine, dopamine, tryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in prostate tumors. The changes appeared to be androgen specific since reintroduction of testosterone restored several of these biochemical parameters virtually to control limits. Chemical sympathectomy induced by 6-hydroxydopamine failed to alter monoamine metabolism; however, the prostatic tumor grown in 6-hydroxydopamine-treated rats showed significantly (32%) less necrosis than those grown in normal animals.     

Biogenic amines in the two-spotted spider mite

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Expression of a costly, plastic secondary sexual trait is correlated with age and condition in a damselfly with two male morphs

Males of the damselfly Mnais costalis Selys (Odonata: Calopterygidae) are morphologically and behaviourally polymorphic, typically existing as clear-winged non-territorial ‘sneaks’ and orange-winged territorial ‘fighters’. The amount of orange pigment in the wing, as measured with a chromameter, varied between individuals, and decreased as the reproductive season progressed. Young individuals maintained in the laboratory on high or low nutrient diets differed in the amount of pigment that developed in the wing. Males in the high nutrient group developed darker wings faster than those in the low nutrient group. Young adults of both sexes and morphs were fed ¹⁴C-radiolabelled tryptophan or tyrosine (precursors of the pigments ommochrome and melanin, respectively). Ommochrome was restricted to the pseudopterostigma of the males of both morphs and was not present in females. The presence of tyrosine in the wing cells of orange males, but not of clear males, indicated that the orange pigment is at least partly constituted from melanin. These data show that at least some pigment levels must be maintained continuously in the wings of orange males, and that maintenance is costly as it is compromised at low nutrient levels.     

Immobilization Decreases Amino Acid Concentrations in Plasma but Maintains or Increases Them in Brain

Immobilization for 2 h significantly decreased plasma concentrations of 13 of 16 amino acids assayed, including the transmitter amine precursors tyrosine and total tryptophan. The level of plasma free tryptophan, however, was increased. Despite the reduced plasma levels, corresponding brain concentrations of many large neutral amino acids (LNAAs) were increased (tryptophan, phenylalanine, valine, leucine, and isoleucine). Brain concentrations of tyrosine and the other amino acids measured were unaltered. The results for the LNAAs were not explained by calculated brain influx rates. Therefore, altered influx kinetics or perhaps altered brain protein metabolism or efflux may be responsible. Comparison of calculated brain influxes and brain concentrations of LNAAs suggests that the rise in level of plasma free tryptophan during immobilization is not responsible for the increase in level of brain tryptophan and that the mechanism responsible for the maintenance of or increase in brain concentrations of the other LNAAs is probably involved. Maintenance of brain concentrations of basic amino acids is explicable by reduced competition for brain uptake.     

Plasma amino acids and sports injuries

Summary. The aim of this study was to explore the relationship between changes in plasma amino acids and the incidence of sports injuries during a soccer season. Fourteen plasma amino acids were assayed at monthly intervals in 12 young soccer players during a whole soccer season. Based on the number and severity of injuries the soccer players were divided into an injury-prone and a non-injury-prone group. The mean plasma level of the amino acid glycine was significantly lower (P=0.009) in the injury-prone group than the other group, while the mean plasma levels of tyrosine, tryptophan and lysine were higher in the injury-prone group during this period (P<0.05). However there were no significant differences in the calculated plasma tryptophan and tyrosine/large neutral amino acids ratios. Significant linear time trends were observed for taurine, ornithine, lysine and the tryptophan/large neutral amino acids ratio.These results indicate that the plasma concentrations of glycine and to a lesser extent those of tyrosine, tryptophan and lysine may be promising peripheral markers for injury-proneness in young soccer players. Whether a role for glycine substitution will be indicative to reduce the occurrence of sports injuries will need to be investigated in future studies.     

Metabolic modulation of redox state confounds fish survival against Vibrio alginolyticus infection

Vibrio alginolyticus threatens both humans and marine animals, but hosts respond to V. alginolyticus infection is not fully understood. Here, functional metabolomics was adopted to investigate the metabolic differences between the dying and surviving zebrafish upon V. alginolyticus infection. Tryptophan was identified as the most crucial metabolite, whose abundance was decreased in the dying group but increased in the survival group as compared to control group without infection. Concurrently, the dying zebrafish displayed excessive immune response and produced higher level of reactive oxygen species (ROS). Interestingly, exogenous tryptophan reverted dying rate through metabolome re-programming, thereby enhancing the survival from V. alginolyticus infection. It is preceded by the following mechanism: tryptophan fluxed into the glycolysis and tricarboxylic acid cycle (TCA cycle), promoted adenosine triphosphate (ATP) production and further increased the generation of NADPH. Meanwhile, tryptophan decreased NADPH oxidation. These together ameliorate ROS, key molecules in excessive immune response. This is further supported by the event that the inhibition of pyruvate metabolism and TCA cycle by inhibitors decreased D. reiro survival. Thus, our data indicate that tryptophan is a key metabolite for the host to fight against V. alginolyticus infection, representing an alternative strategy to treat bacterial infection in an antibiotic-independent way.     

Differential effects of amino acids on the period of the circadian rhythm from the Aplysia eye

The effect of continuous treatments of single L -amino acids (0.1mM) on the free running rhythm from the isolated Aplysia eye was examined. A variation in the change in free running period produced by different amino acids was observed. Two well-known precursors of neurotransmitter (tyrosine, tryptophan) had the largest effects. These amino acids lengthened the period ca. 1.7 h. Another group of amino acids (alanine, threonine, proline) lengthened the period by about 1 h. Smaller effects were produced by aspartic acid and leucine and no effects were caused by lysine, glycine, valine, and serine. Phenylalanine may shorten the period a small amount. Glucose (5mM) lengthens the period a small amount (0.4 h), decreases the effect of tyrosine somewhat, and has no effect on the lengthening of the period produced by tryptophan. Three amino acids not involved in protein synthesis (ornithine, β-alanine, citrulline) had at most small effects on the free running period. Also, D -tryptophan lengthened the period by 0.6 h but the effect of D -tryptophan was considerably smaller than the effect of L -tryptophan. A few of the amino acids had small short-term effects on spike rate and longer-term effects on the amplitudes of the rhythms but these effects did not correlate with the effects of the amino acids on the free running period. Though continuous treatments of certain amino acids lengthened the periods, shorter treatments (tryptophan, 6 h) did not phase-shift the rhythm. Since eyes maintained in a commonly used culture medium have longer periods than eyes in a simple seawater medium, the amino acids of the culture medium must be responsible, at least in part, for the lengthening effect of the culture medium. The mechanism of action of the amino acids is unknown. The magnitude of the effects did not correlate with physical-chemical properties of the amino acids nor with whether the amino acids were “essential” or “nonessential.” The effects of the amino acids may be mediated by their effects on neurotransmitter and/or protein synthesis.     

Metabolism of tryptophan,methionine and arginine in <Emphasis Type="Italic">Diplodus sargus</Emphasis> larvae fed rotifers: effect of amino acid supplementation

Dietary amino acids imbalances have been described when fish larvae are fed rotifers, what may lead to a reduction in growth rate. The tube-feeding technique can be used to assess the effect of free amino acid short term supplementation. In this study supplementation of tryptophan, methionine and arginine were tested in Diplodus sargus. Single crystalline ¹⁴C amino acids as well as a mix of ¹⁴C amino acids were used as tracers to compare results of individual amino acids metabolism with the average of all amino acids. The results show low absorption efficiencies for tryptophan (70%) and arginine (80%) and similar absorption for methionine (90%) when compared with the average of all amino acids. Supplementation of these amino acids seems to be viable but it did not result in higher retention compared to the amino acid mix. This means that tryptophan, methionine and arginine are probably not the limiting amino acid when Diplodus sargus larvae are fed rotifers. However, supplementation in these IAA may be required for their roles as precursors of important molecules other than proteins, in order to improve larval quality and/or performance.     

The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa

Summary Eighteen auxotrophs of Pseudomonas aeruginosa requiring l-tryptophan for growth were isolated following nitrosoguanidine mutagenesis. Mutant blocks for each step of tryptophan biosynthesis were identified by enzymological assay. A regulatory mutant was characterized which was simultaneously constitutive for the gene products of trpA, trpB and trpD. Another class of regulatory mutant appears to synthesize tryptophan synthetase (i.e., trpE and trpF subunits) constitutively. The results implicate three control entities in the pathway of tryptophan biosynthesis: (i) The gene products of trpA, trpB and trpD are repressible by tryptophan, the range of enzyme specific activity varying at least fifty-fold. (ii) No regulation of the trpC gene product could be demonstrated, indicating that its synthesis is constitutive. (iii) The gene products of rpE and trpF are inducible by indoleglycerol 3-phosphate; the magnitude of induction can exceed 100-fold. These results together with some genetic data indicate a general similarity in gene-enzyme relationships between P. aeruginosa and P. putida. A number of specific differences that distinguish the two species are noted.A mutant blocked in the common pathway of aromatic biosynthesis was used to prove that enzymes of tryptophan biosynthesis other than tryptophan synthetase are not inducible by precursors of the common pathway such as chorismate. It is concluded that the concentration of tryptophan that signals total repression of the gene products of trpA, trpB and trpD is lower than the concentrations necessary for maximal feedback inhibition of anthranilate synthetase and for abolition of the induction of tryptophan synthetase.     

Regulation of chorismate mutase activity of various yeast species by aromatic amino acids

The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species. All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol. The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan. The tryphtophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 M (Yarrowia lipolytica). Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine. The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone. The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan. Six enzyme sources were not inhibited and theY. lipolytica chorismate mutase was slightly activated by both amino acids.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

Regulation of chemoautotrophic metabolism

Summary Incorporation of ¹⁴C-phenylalanine by T. neapolitanus was inhibited competitively by relatively low concentrations of glycine, serine, alanine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, threonine, and methionine (Group I amino acids), but not greatly depressed by aspartate, glutamate, lysine, arginine, cysteine (Group II amino acids) and proline at similar concentrations. Group I acids competed with each other for incorporation but were little affected by Group II acids. Similarly Group I acids little depressed the incorporation of Group II acids, among which, however, some mutual inhibition occurred. Incorporation of proline was depressed by both Group I and II acids. Two main permeation mechanisms are proposed, one transporting Group I acids, the other Group II acids, but some overlapping of function probably occurs. Proline may be transported by a third permease, which is subject to inhibition by both Group I and II acids. T. concretivorus also has a common transport mechanism for some amino acids. Less interaction between amino acids was found using two heterotrophic pseudomonads.Exogenous phenylalanine inhibited both the biosynthesis and the uptake of tyrosine and tryptophan by T. neapolitanus. High phenylalanine concentrations depressed the assimilation of ¹⁴C-labelled tyrosine and tryptophan less than low ones, suggesting that the bacteria developed a requirement for external tyrosine and tryptophan when exposed to highly inhibitory concentrations of phenylalanine.     

Raman studies of the crystalline, solution, and alkaline-denatured states of beta-lactoglobulin.

The Raman spectra of β-lactoglobulin in the crystalline, freeze-dried, and solution states are compared. The spectra of the freeze-dried and crystalline proteins were practically identical. The conformationally sensitive amide III line appearing at 1242 cm^?1 increased in intensity 30% upon dissolution of the protein in water which is interpreted as a conformational change in the disordered chains of the protein. This result appears to be a phenomenon for globular proteins containing a large disordered chain fraction. The alkaline denaturation of β-lactoglobulin was studied. When the pH was increased from 6.0 to 11.0, the amide III line shifted from 1242 to 1246 cm^?1, broadened, and decreased in intensity. This is consistent with the conversion of β-sheet regions in β-lactoglobulin to the disordered conformation, as has been proposed by other investigators. At pH 13.5 the amide III shifts to 1257 cm^?1 characteristic of a completely disordered protein, indicating that any remaining “core” of β-sheet has been randomized. Several changes in the intensities of the tyrosine and tryptophan vibrations accompany the denaturation. As the pH is increased from 6.0 (native state) to 11.0 (denatured state) the intensity ratio of two tyrosine ring vibrations, I₈₅₅ cm^?1/I₈₃₀ cm^?1, decreases from 1.0:0.9 to 1.0:1.3. The same ratio for a copolymer consisting of 95% glutamic acid and 5% tyrosine at pH 7.0, where the polymer forms a random coil exposing the tyrosine to the aqueous environment, is 1.0:0.62. This ratio more closely resembles that corresponding to β-lactoglobulin at pH 6.0 (native state) than pH 11.0 (denatured state) suggesting that the average tyrosine in the denatured state may be in a more hydrophobic environment than in the native state. A time-dependent polymerization of the denatured protein reported by other investigators and observed by us may account for the change in the tyrosine environment. A tryptophan vibration appearing at 833 cm^?1 in the spectrum of the native state becomes weak as the pH is increased to 11.0. The intensity of this line may also reflect the local environment of the tryptophan residue.     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.