Inhibition of yeast tRNATrp aminoacylation by 4-methyltryptophan

The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNA^Trp). The mode of inhibition is competitive and the analogue is not charged onto tRNA^Trp. Thus 4-methyltryptophan application depletes the cells from charged tRNA^Trp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.     

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

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.     

Relationship Between Plasma and Brain Tryptophan in Pigs During Experimental Hepatic Coma Before and After Hernodialysis with Selective Membranes

Experimental acute liver ischemia in pigs induces an increment in plasma free tryptophan with decreased total tryptophan. Brain tryptophan is elevated in all brain areas. A slight, but significant increase of brain serotonin is demonstrated in the striatum only, while 5-HIAA (5-hydroxyindoleacetic acid) is significantly lower in the hypothalamus. Other brain areas do not show significant changes in serotonin and 5-HIAA levels. Neither the high plasma free tryptophan levels, nor the decreased sum of neutral competitive amino acids are consistent with such an elevation of brain tryptophan. Hemodialysis was carried out with two different kinds of membranes: cuprophan (with an efficient removal of molecules up to molecular weight 1300) and AN 69 polyacrylonitrile (efficient removal up to 15,000). Ammonia and aminoacid clearance are similar for both membranes. After AN 69, plasmatic free tryptophan and brain tryptophan are lower than after liver devascularization, but still higher than normal. Serotonin significantly increases in the cortex, midbrain and hypothalamus without concomitant rise of 5-HIAA levels. After cuprophan hemodialysis, plasma total tryptophan is lower than in normal and even comatose animals, whereas free tryptophan is normal. Intracerebral tryptophan is similar to AN 69 dialysed animals, but in the hypothalamus it is similar to nondialysed animals. Brain serotonin levels are not modified. 5-HIAA decreases in the hypothalamus. This finding suggests that middle molecules (which are not cleared out with cuprophan hemodialysis) are involved in the intracerebral transfer of tryptophan and the metabolism of serotonin, mainly in the hypothalamus.     

Elevated Monoamine Levels in the Cerebral Hemispheres of Microencephlic Rats Treated Prenatally with Methylazoxymethanol or Cytosine Arabinoside

Abstract: Methylazoxymethanol (MAM) injection to rats on day 15 of gestation caused a significant rise in monoamine concentrations (1.6, 2.0, and 2.8 times the control value for serotonin, norepinephrine, and dopamine, respectively) accompanying a decrease in the brain weight and DNA content in the cerebral hemispheres of the offspring at 3 months of age; in the brain stem, these changes were much smaller. Similar change of monoamine concentrations was observed in cytosine arabinoside-induced microencephaly. The decrease of DNA content and the elevation of monoamine levels were lower with MAM injection on day 15, 13, or 17 of gestation (in that order). Serotonin content of the MAM-treated cerebral hemispheres was already 50% higher than the control immediately after birth. The activity of tryptophan hydroxylase in the MAM-treated cerebrum was 1.6 times the control value, with no change in the brain stem, while the concentration of tryptophan in the brain and plasma was equal to the control value, suggesting an important role played by this enzyme in the elevation of serotonin content. Although the marked decrease of DNA content in the cerebral hemispheres of MAM-treated rats indicates a loss of cerebral cells due to prenatal MAM poisoning, the kind of cells destroyed remain to be studied. That the remaining neurons, axons, and oligodendroglia were intact was suggested by the normal activity of CNPase.     

Sequestration of arginine by polyphosphate in vacuoles of yeast (Saccharomyces cerevisiae)

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both , -elimination reactions (using tryptophan, serine, cysteine and S-methyl-cysteine as substrates) and -replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.Nonstandard Abbreviations PLP pyridoxal 5-phosphate - TPase tryptophanase - TSase tryptophan synthase - DHase dehydratase - TCA tricarboxylic acid - BSA bovine serum albumin Preliminary reports of this work have been presented (M. J. Klug and R. D. DeMoss, Bacteriol. Proc. 1971, p. 132; D. D. Whitt and R. D. DeMoss, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, p. 148)     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Tryptophan-based fluorophores for studying protein conformational changes

With the continuing interest in deciphering the interplay between protein function and conformational changes, small fluorescence probes will be especially useful for tracking changes in the crowded protein interior space. Presently, we describe the potential utility of six unnatural amino acid fluorescence donors structurally related to tryptophan and show how they can be efficiently incorporated into a protein as fluorescence probes. We also examine the various photophysical properties of the new Trp analogues, which are significantly redshifted in their fluorescence spectra relative to tryptophan. In general, the Trp analogues were well tolerated when inserted into Escherichia coli DHFR, and did not perturb enzyme activity, although substitution for Trp22 did result in a diminution in DHFR activity. Further, it was demonstrated that D and E at position 37 formed efficient FRET pairs with acridon-2-ylalanine (Acd) at position 17. The same was also true for a DHFR construct containing E at position 79 and Acd at position 17. Together, these findings demonstrate that these tryptophan analogues can be introduced into DHFR with minimal disruption of function, and that they can be employed for the selective study of targeted conformational changes in proteins, even in the presence of unmodified tryptophans.     

宽泛底物谱色氨酸脱羧酶体外合成卤代色胺衍生物和血清素

【背景】色氨酸脱羧酶在自然界有高度特异性,行使催化色氨酸为色胺的功能。一个色氨酸脱羧酶BaTDC参与南海海绵共生菌Bacillus atrophaeus C89次级代谢产物bacillamides的生物合成过程。【目的】探究BaTDC酶学特征和底物谱,建立体外合成色胺衍生物的方法。【方法】通过构建系统发育树揭示BaTDC在进化中的地位。在温度梯度和pH梯度下进行酶反应,利用不同的色氨酸衍生物为底物,通过HPLC和UPLC-MS检测酶反应过程,表征BaTDC活性。【结果】系统发育分析显示BaTDC与肠道菌Ruminococcus gnavus亲缘关系相近。纯化重组BaTDC的最适温度为40?45 °C,最适pH值为8.0。BaTDC可以催化羟代色氨酸和卤代色氨酸包括4-氟色氨酸和5,6,7-氯色氨酸及4-溴色氨酸,得到相应的卤代色胺衍生物和血清素。【结论】本研究分析了BaTDC的特性,发现BaTDC表现出宽泛的底物耐受性,可为前体喂养或定向生物合成新型药用色胺衍生物和下游复杂天然产物奠定基础。     

Evolutionary patterns of the mitochondrial genome in the Moorish gecko, Tarentola mauritanica

A previous study on the evolutionary patterns of Tarentola mauritanica demonstrated that low levels of mitochondrial diversity observed in the European populations relative to nuclear markers were consistent with a selective sweep hypothesis. In order to unravel the mitochondrial evolutionary history in this European population and two other lineages of T. mauritanica (Iberian and North African clades), variation within 22 nearly complete mitogenomes was analyzed. Surprisingly, each clade seems to have a distinct evolutionary history; with both the European and Iberian clades presenting a decrease of polymorphism, which in the former is consistent with departure from neutrality of the mtDNA (positive or background selection), but in the latter seems to be the result of a bottleneck after a population expansion. The pattern exhibited by the North African clade seems to be a consequence of adaptation to certain mtDNA variants by positive selection.