Clustering of mutations affecting central pathway enzymes of carbohydrate catabolism in Pseudomonas aeruginosa

Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of 24, 3.5, and 1.8 microM. Transport of these amino acids was optimum at pH 7.5 and 25 degrees C for phenylalanine and pH 8.0 and 35 degrees C for tyrosine and tryptophan. Crossed inhibitions were all noncompetitive. The only marked stereospecificity was for the L form of phenylalanine. Transport was almost totally inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were much more inhibitory for tryptophan transport than for transport of the other two aromatic amino acids.     

Formation of aromatic amino acid pools in Escherichia coli K-12

Phenylalanine, tyrosine, and tryptophan were taken up into cells of Escherichia coli K-12 by a general aromatic transport system. Apparent Michaelis constants for the three amino acids were 4.7 x 10(-7), 5.7 x 10(-7), and 4.0 x 10(-7)m, respectively. High concentrations (> 0.1 mm) of histidine, leucine, methionine, alanine, cysteine, and aspartic acid also had an affinity for this system. Mutants lacking the general aromatic transport system were resistant to p-fluorophenylalanine, beta-2-thienylalanine, and 5-methyltryptophan. They mapped at a locus, aroP, between leu and pan on the chromosome, being 30% cotransducible with leu and 43% cotransducible with pan. Phenylalanine, tyrosine, and tryptophan were also transported by three specific transport systems. The apparent Michaelis constants of these systems were 2.0 x 10(-6), 2.2 x 10(-6), and 3.0 x 10(-6)m, respectively. An external energy source, such as glucose, was not required for activity of either general or specific aromatic transport systems. Azide and 2,4-dinitrophenol, however, inhibited all aromatic transport, indicating that energy production is necessary. Between 80 and 90% of the trichloroacetic acid-soluble pool formed from a particular exogenous aromatic amino acid was generated by the general aromatic transport system. This contribution was abolished when uptake was inhibited by competition by the other aromatic amino acids or by mutation in aroP. Incorporation of the former amino acid into protein was not affected by the reduction in its pool size, indicating that the general aromatic transport system is not essential for the supply of external aromatic amino acids to protein synthesis.     

Transport of Aromatic Amino Acids by Pseudomonas aeruginosa

Kinetic studies of the transport of aromatic amino acids by Pseudomonas aeruginosa revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of P. aeruginosa at a concentration which was 10 times greater than that of tryptophan.     

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.     

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.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

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.     

Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus

Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.     

Transport of 14C-L-glycine by the haustorial mycoparasite Tieghemiomyces parasiticus in axenic culture.

Transport of L-glycine by the haustorial mycoparasite Tieghemiomyces parasiticus grown in axenic culture was investigated using endogenously metabolizing cells. Transport occurred against a concentration gradient and required the expenditure of energy supplied by metabolic reactions that were inhibited by catalytic amounts of sodium azide and 2,4-dinitrophenol. The process exhibited both pH and temperature dependence with optima at pH 4 and 35 degrees C, respectively. The energy of activation and Q10 for transport were calculated to be 18,227 cal/mole and 2.82 respectively, between 20 and 30 degrees C. The kinetics of the system were consistent with a mechanism of transport that depended upon a finite number of sites on the cell surface and had a Kt of 1.46 x 10(-5) M. The system for L-glycine transport lacked sterospecificity and was most inhibited in the presence of other neutral amino acids while less inhibition was observed in the presence of the acidic and basic amino acids. These results suggest that L-glycine transport in this mycoparasite occurs by an energy dependent, protein-mediated process characteristic of active transport.     

Maintenance and exchange of the aromatic amino acid pool in Escherichia coli

The pool of phenylalanine, tyrosine, and tryptophan is formed in Escherichia coli K-12 by a general aromatic transport system [Michaelis constant (K(m)) for each amino acid approximately 5 x 10(-7)m] and three further transport systems each specific for a single aromatic amino acid (K(m) for each amino acid approximately 2 x 10(-6)m, reference 3). When the external concentration of a particular aromatic amino acid is saturating for both classes of transport system, the free amino acid pool is supplied with external amino acid by both systems. Blocking the general transport system reduces the pool size by 80 to 90% but does not interfere with the supply of the amino acid to protein synthesis. If, however, the external concentration is too low to saturate specific transport, blocking general transport inhibits the incorporation of external amino acid into protein by about 75%. It is concluded that the amino acids transported by either class of transport system can be used for protein synthesis. Dilution of the external amino acid or deprivation of energy causes efflux of the aromatic pool. These results and rapid exchange observed between pool amino acid and external amino acids indicate that the aromatic pool circulates rapidly between the inside and the outside of the cell. Evidence is presented that this exchange is mediated by the aromatic transport systems. Mutation of aroP (a gene specifying general aromatic transport) inhibits exit and exchange of the small pool generated by specific transport. These findings are discussed and a simple physiological model of aromatic pool formation, and exchange, is proposed.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Effect of glyphosate on carrot and tobacco cells

The growth of suspension-cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N-[phosphonomethyl]glycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosate-induced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added.     

Thermal Degradation of Aromatic Amino Acids

Aromatic amino acids (phenylalanine, tyrosine and tryptophan) were heated at 300°C under nitrogen and volatile compounds generated were examined. Twelve compounds in which many of them have aromatic rings were identified in the volatiles from thermal degradation of phenylalanine. Tyrosine and tryptophan produced some phenols and indoles, respectively, besides several compounds. Formation mechanisms of some compounds were also discussed.     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

Inhibition of growth of cells in culture by L-phenylalanine as a model system for the analysis of phenylketonuria. I. Amino acid antagonism and the inhibition of protein synthesis

Phenylalanine in high concentrations inhibits the growth of mouse A9 cells. Protein synthesis is inhibited earlier and more severely than RNA or DNA synthesis. Phenylalanine inhibits the uptake and decreases the intracellular pool of several amino acids. Certain amino acids added in excess reverse the phenylalanine inhibition. The strongest reversing amino acids appear to function by excluding phenylalanine. The phenylalanine inhibition does not appear to be due to a deficiency of any amino acid, but to the high intracellular phenylalanine concentration and/or an amino acid imbalance resulting from the large ratio of phenylalanine to other amino acids.     

Transport of benzenoid amino acids by system T and four broad scope systems in preimplantation mouse conceptuses

We have studied transport of L-tryptophan, L-tyrosine and L-phenylalanine as factors contributing to homeostasis of these amino acids in preimplantation mouse conceptuses. Benzenoid amino acids were transported by the Na(+)-independent systems L and b0,+ in 1-cell conceptuses, and by these systems plus the Na(+)-dependent systems B0,+ and B in blastocysts. In addition, a component of Na(+)-independent tryptophan, tyrosine and phenylalanine transport in 1-cell and 2-cell conceptuses and in blastocysts resisted inhibition by L-leucine. The latter component of transport not only preferred benzenoid amino acids and in particular tryptophan as substrates, but it also was inhibited strongly and competitively by alpha-N-methyl-L-tryptophan. The leucine-resistant component of tryptophan transport also was inhibited strongly by N-ethylmaleimide and D-tryptophan, and it appeared to be inhibited weakly by 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid (BCO) but not by other amino acids tested as inhibitors. By these criteria, the leucine-resistant component of transport of benzenoid amino acids resembled system T in human red blood cells and rat hepatocytes. It is not entirely clear why preimplantation blastocysts have five good systems for transport of tryptophan. It is possible, however, that tryptophan homeostasis is particularly important during preimplantation development since it has been shown elsewhere that tryptophan availability in blood increases within one day after rat eggs are fertilized.